Novel flavivirus antigens

ABSTRACT

The invention provides polynucleotides and polypeptides encoded therefrom having advantageous properties, including an ability to induce an immune response to flaviviruses. The polypeptides and polynucleotides of the invention are useful in methods of inducing immune response against flaviviruses, including dengue viruses. Compositions and methods for utilizing polynucleotides and polypeptides of the invention are also provided.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and benefit of provisionalU.S. Patent Application Serial No. 60,360,030, filed Feb. 26, 2002, thedisclosure of which is incorporated herein by reference in its entiretyfor all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was developed in part with Government support by agrant from the Defense Advanced Research Projects Agency (DARPA) (GrantNo. N65236-99-1-5421). The Government may have certain rights in thisinvention.

COPYRIGHT NOTIFICATION

[0003] Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion ofthis disclosure contains material which is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or patent disclosure, asit appears in the Patent and Trademark Office patent file or records,but otherwise reserves all copyright rights whatsoever.

FIELD OF THE INVENTION

[0004] This invention pertains generally to polypeptides that induce animmune response against one or more dengue viruses and/or otherflaviviruses, polynucleotides encoding such polypeptides, methods ofmaking and using such polypeptides, polynucleotides, and diagnosticassays employing such polypeptide and polynucleotides.

BACKGROUND OF THE INVENTION

[0005] Viruses of the Flavivirus genus are positive-sense,single-stranded RNA viruses of the Flaviviridae family, many of whichare responsible for disease in humans and other mammals. Examples offlaviviruses include Tick-borne encephalitis virus, Japaneseencephalitis virus, Yellow Fever virus, St. Louis encephalitis virus,hepatitis C virus, and West Nile viruses. Flaviviruses of the Flavivirusgenus generally comprise three major mature structural proteins: anenvelope (E) protein, a capsid (C) protein, and a membrane (M) protein.The M protein is usually formed as a proteolytic fragment of apre-membrane (prM) protein. See, e.g., FIELDS VIROLOGY 997-998, RavenPress, Ltd., New York (D. M. Knipe et al. eds., 4th ed., 2001), 996(hereinafter “FIELDS VIROLOGY”), and the ENCYCLOPEDIA OF VIROLOGY (R. G.Webster et al. eds., Academic Press, 2nd ed., 1999), each of which isincorporated herein by reference in its entirety for all purposes.

[0006] Dengue (DEN) viruses are known among flaviviruses as agents ofdisease in humans. Dengue viruses comprise four known distinct, butantigenically related serotypes, named Dengue-1 (DEN-1 or Den-1),Dengue-2 (DEN-2 or Den-2), Dengue-3 (DEN-3 or Den-3), and Dengue-4(DEN-4 or Den-4). Dengue virus particles are typically spherical andinclude a dense core surrounded by a lipid bilayer. FIELDS VIROLOGY,supra.

[0007] The genome of a dengue virus, like other flaviviruses, typicallycomprises a single-stranded positive RNA polynucleotide. FIELDSVIROLOGY, supra, at 997. The genomic RNA serves as the messenger RNA fortranslation of one long open reading frame (ORF) as a large polyprotein,which is processed co-translationally and post-translationally bycellular proteases and a virally encoded protease into a number ofprotein products. Id. Such products include structural proteins andnon-structural proteins. A portion of the N-terminal of the genomeencodes the structural proteins—the C protein, prM (pre-membrane)protein, and E protein—in the following order: C-prM-E. Id. at 998. TheC-terminus of the C protein includes a hydrophobic domain that functionsas a signal sequence for translocation of the prM protein into the lumenof the endoplasmic reticulum. Id. at 998-999. The prM protein issubsequently cleaved to form the structural M protein, a smallstructural protein derived from the C-terminal portion of prM, and thepredominantly hydrophilic N-terminal “pr” segment, which is secretedinto the extracellular medium. Id. at 999. The E protein is a membraneprotein, the C-terminal portion of which includes transmembrane domainsthat anchor the E protein to the cell membrane and act as signalsequence for translocation of non-structural proteins. Id. The E proteinis the major surface protein of the virus particle and is believed to bethe most immunogenic component of the viral particle. The E proteinlikely interacts with viral receptors, and antibodies that neutralizeinfectivity of the virus usually recognize the E protein. Id. at 996.The M and E proteins have C-terminal membrane spanning segments thatserve to anchor these proteins to the membrane. Id. at 998.

[0008] Dengue viruses are primarily transmitted to humans through themosquito Aedes aegypti. There is a significant threat of dengueinfection to people living in or visiting tropical areas. Indeed, anestimated 2.5 billion people live in areas at risk for transmission andover 100 million humans are infected each year. Infection with denguevirus is estimated to kill approximately 20-25,000 children each year.

[0009] An initial dengue virus infection is clinically manifested formost of the cases by dengue fever (DF), which is a self-limited fibrilillness. Although rarely fatal, DF is characterized by often-severedisseminated body pain, headache, fever, rash, lymphadenopathy andleukopenia. Subsequent infection with a heterologous Dengue virus canlead to the much more severe to fatal disease of dengue hemorrhagicfever (DHF) or dengue shock syndrome (DSS). It is hypothesized that thepresence of antibodies to the serotype causing the primary infectionenhances the infection by a heterologous serotype in secondaryinfections. This phenomenon is referred to as antibody-dependentenhancement (ADE) of the disease.

[0010] Effective diagnosis of dengue virus is often problematic. Allfour dengue virus serotypes can be prevalent in one local area and it istherefore important to test a subject's serum samples simultaneouslyagainst all 4 dengue virus serotypes.

[0011] There is currently no specific treatment for dengue virusinfections. Although the development of dengue virus vaccines has beenongoing for the past 50 years, no successful vaccine to dengue virus hasbeen produced and no licensed dengue virus vaccine is yet available. Amajor challenge is to generate a tetravalent vaccine that inducesneutralizing antibodies against all four strains of dengue to avoid ADEwhen the individual encounters viruses of two or more differentserotypes. Vaccine strategies using a mixture of DEN 1-4 attenuatedviruses have been largely unsuccessful, because the antigens from onetype will tend to dominate or “mask” the others, producing an incompleteimmune response across the four types.

[0012] There remains a need for molecules, compositions and methods foreffectively diagnosing one or more dengue viruses, inducing, enhancing,or promoting an immune response to flaviviruses, particularly dengueviruses, and preferably to all four dengue virus serotypes, andprophylactically or therapeutically treating disorders or diseasesrelated to one or more such viruses. The present invention provides suchmolecules, methods and compositions. These and other advantages of theinvention, as well as additional inventive features, will be apparentfrom the description of the invention provided herein.

SUMMARY OF THE INVENTION

[0013] The invention provides novel recombinant, synthetic, mutant,and/or isolated polypeptides as described herein, fusion proteinscomprising such polypeptides, and nucleic acids encoding suchpolypeptides and/or proteins, that are useful in promoting (e.g.,inducing and/or enhancing) an immune response to one or moreflaviviruses, particularly one or more dengue viruses, and detecting ordiagnosing the presence of anti-Flaviviridae virus antibodies (e.g.,anti-flavivirus antibodies) against at least one virus of theFlaviviridae family (e.g., members of the Flavivirus genus), and/oranti-dengue virus antibodies against at least one dengue virus in abiological sample.

[0014] In one aspect, for example, the invention provides recombinant,synthetic, mutant, and/or isolated polypeptides that each comprise anamino acid sequence that has at least about 80%, 83%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identity to an amino acid sequence comprising a truncatedrecombinant, synthetic or mutant dengue virus envelope (E) proteinpolypeptide of the invention, such as at least one of any of SEQ IDNOS:1-49 and 153-155. Some such polypeptides induce an immune responsein a subject, e.g., mammal or population of mammalian cells against atleast one dengue virus, or dengue virus antigen, of at least oneserotype selected from the group of dengue-1, dengue-2, dengue-3, anddengue-4 in vitro or ex vivo in cells and/or in vivo in a subject orcells or tissue thereof. Such a recombinant dengue E protein polypeptideis “truncated,” since by comparison with the full length sequence of awild-type (WT) dengue virus E protein, it lacks one or more amino acidresidues from the C terminal of the full length E protein sequence.Usually, a truncated E protein lacks from about 3%, 5%, 10%, 15%, or 20%to about 25% of the C terminal amino acid residues of the full length Eprotein. Each such polypeptide comprises an immunogenic or antigenicamino acid sequence that is capable of inducing an immune responseagainst one or more dengue viruses, or virus-like particle (VLP) orantigen thereof, of one or more, preferably multiple (e.g., 2, 3, or 4)serotypes. Some such polypeptides induce an immune response to all 4dengue virus serotypes (a tetravalent immune response) or antigens, whenexpressed in, or delivered to, an animal or animal cell. Particularpolypeptides having such characteristics advantageously are capable ofinducing neutralizing antibodies against dengue viruses of multipledengue virus serotypes upon expression in, or delivery to, an animal oranimal cell(s), and, preferably, are able to induce a protective immuneresponse against at least one of the four dengue virus serotypes in asubject, e.g., mammal, such as a primate or a human. Preferably, therecombinant polypeptide induces a protective immune response against allfour dengue virus serotypes in a subject, e.g., mammal.

[0015] Such truncated E protein polypeptides of the invention induce animmune response in a subject against at least one dengue virus, or VLPor antigen thereof, of each of at least one, two, three, or fourserotypes selected from the group of dengue-1, dengue-2, dengue-3, anddengue-4 that is about equal to or greater than an immune responseinduced in the subject against the at least one dengue virus of each ofthe at least one, two, three or four serotypes, or VLP or antigenthereof, by a wild-type truncated E protein of each said at least onedengue virus of each of the one, two, three or four serotypes,respectively, wherein said wild-type truncated E protein has an aminoacid sequence length substantially equivalent to that of therecombinant, synthetic, or mutant polypeptide of the invention.

[0016] Each such recombinant, synthetic, or mutant polypeptides inducesproduction of one or more types of antibodies that bind to at least onedengue virus of each of at least one, two, three or four serotypes. Inone aspect of the invention, some such polypeptides induce production ofa number or population of antibodies that bind to at least one denguevirus of each of at least one, two, three or four serotypes that isabout equal to or greater than the number induced by a wild-typetruncated E protein of the at least one dengue virus of each of the atleast one, two, three or four serotypes, respectively.

[0017] Each such recombinant, synthetic, or mutant polypeptide of theinvention induces or produces a titer of neutralizing antibodies againstat least one dengue virus of each of at least one, two, three or fourserotypes. Further, some such polypeptides induce or produce a titer ofneutralizing antibodies against at least one dengue virus of each of atleast one, two, three, or four serotypes that is about equal to orgreater than a titer of neutralizing antibodies induced or producedagainst the at least one dengue virus of each of the at least one, two,three, or four serotypes by a wild-type truncated E protein of the atleast one dengue virus of each of the at least one, two, three or fourserotypes, respectively, wherein each said wild-type truncated E proteinis selected from the group of SEQ ID NOS:338-341.

[0018] Some such recombinant, synthetic, or mutant polypeptides furthercomprise: (a) an amino acid sequence of at least about 150 amino acidresidues that has at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acidsequence identity to at least one of SEQ ID NOS:117-126 fused to theN-terminus of the amino acid sequence of the recombinant or syntheticpolypeptide; (b) an amino acid sequence of at least about 40 amino acidresidues that has at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acidsequence identity to at least one of SEQ ID NOS:127-136 fused to theC-terminus of the amino acid sequence of the recombinant or syntheticpolypeptide; or (c) the amino acid sequence of (a) and the amino acidsequence of (b).

[0019] The invention also provides a recombinant, synthetic, or mutanttruncated or non-truncated dengue virus envelope protein, wherein therecombinant or synthetic polypeptide is encoded a nucleic acidcomprising a polynucleotide sequence selected from the group of: (a) apolynucleotide sequence having at least about 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or morenucleotide sequence identity to at least one polynucleotide sequenceselected from the group of SEQ ID NOS:285-330 or a complementarypolynucleotide sequence thereof; (b) a RNA polynucleotide sequencecomprising a DNA sequence selected from the group of SEQ ID NOS:285-330in which all of the thymine nucleotide residues in the DNA sequence arereplaced with uracil nucleotide residues or a complementary RNApolynucleotide sequence thereof; (c) a RNA polynucleotide sequence thathas at least about 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% nucleotide sequence identity to at least one RNApolynucleotide sequence of (b) or a complementary RNA polynucleotidesequence thereof; (d) a polynucleotide sequence that hybridizes under atleast stringent conditions over substantially the entire length of apolynucleotide sequence of (a)-(c); (e) a polynucleotide sequence whichwould hybridize under at least stringent conditions over substantiallythe entire length of a polynucleotide sequence of any of (a)-(d) but forthe degeneracy of the genetic code; and (f) a polynucleotide sequencethat possesses any combination of the features of the polynucleotidesequences of (a)-(e); or a complementary sequence of any thereof.

[0020] In another aspect, the invention provides isolated, recombinant,synthetic, or mutant polypeptides that each comprise an amino acidsequence that has at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more amino acidsequence identity to the amino acid sequence of at least one of SEQ IDNOS:65-116. Each such polypeptide typically comprises a recombinant,synthetic, or mutant dengue virus antigen comprising a PRM15/truncated Epolypeptide. Such PRM15/truncated E protein polypeptide comprises afirst amino acid sequence comprising a methionine at the N terminusfused to a second recombinant, mutant, or synthetic amino acid sequencecomprising about 15 amino acid residues that may correspond to about thelast 15 amino acids of the C terminal of a recombinant, synthetic ormutant dengue virus prM protein. The C terminus of the second amino acidsequence is fused in turn to the N terminus of a 3rd sequence whichcomprises a recombinant, mutant or synthetic truncated dengue virus Eprotein. The truncated E protein is deemed “truncated,” since bycomparison with a WT full length dengue virus E protein, it lacks one ormore amino acids at the C terminus of the E protein. A truncated Eprotein may comprise about 75, 80, 85 or 90% E protein, said 75, 80, 85,or 90% E protein representing a portion of the E protein that comprisesabout 75, 80, 85, or 90%, respectively, of its length starting fromamino acid 1 at its N-terminus. Such recombinant or syntheticPRM15/truncated E polypeptide induces an immune response against atleast one dengue virus of at least one serotype, or VLP or antigenthereof.

[0021] Some such PRM15/truncated E polypeptides induce an immuneresponse in a subject against at least one dengue virus of one serotypethat is about equal to or greater than the immune response induced inthe mammal against the same dengue virus of the same serotype by aPRM15/truncated E protein derived from the dengue virus serotype. Somesuch PRM15/truncated E polypeptides induce an immune response in asubject against each of at least 2 dengue viruses of 2 differentserotypes that is about equal to or greater than the immune responseinduced in the subject against each of these 2 dengue viruses ofdifferent serotypes by a PRM15/truncated E protein made from either ofthe two serotypes. Some such PRM15/truncated E polypeptides each inducean immune response against each of at least three dengue viruses ofthree different serotypes that is about equal to or greater than theimmune response induced in such cell against these three dengue virusesof three different serotypes by a PRM15/truncated E protein derived fromany of the three serotypes. Some such PRM15/truncated E polypeptidesinduce an immune response in a subject against each of at least 4 dengueviruses of four different serotypes that is about equal to or greaterthan the immune response induced in such cell against these 4 dengueviruses of four different serotypes by a PRM15/truncated E proteinderived from any of the four serotypes. In one aspect, aWTPRM15/truncated E protein polypeptide is selected from SEQ IDNOS:149-152.

[0022] PRM15/truncated E polypeptides are capable of inducing productionof a population of antibodies comprising one or more types of antibodiesthat bind to at least one dengue virus of each of the at least 1, 2, 3or 4 serotypes. Some such polypeptides induce the production of apopulation or number of antibodies that bind to at least one denguevirus of each of at least one, two, three or four serotypes that isabout equal to or greater than is induced by the wild-typePRM15/truncated E protein polypeptide of each of the at least one, two,three, or four serotypes.

[0023] Furthermore, recombinant, synthetic, or mutant PRM15/truncated Epolypeptides induce the production of a population of antibodies thatbind more specifically to at least one dengue virus of each of at leastone, two, three or four serotypes than is induced by the wild-typePRM15/truncated E polypeptide of the at least one dengue virus of eachof the at least one, two, three, or four serotypes, respectively,wherein each said wild-type PRM15/truncated E polypeptide is selectedfrom SEQ ID NOS:149-152.

[0024] In one aspect, recombinant, synthetic, or mutant PRM15/truncatedE polypeptides induce the production of a titer of neutralizingantibodies against at least one dengue virus of each of at least one,two, three, or four serotypes. In a particular aspect, some of theserecombinant, synthetic, or mutant PRM15/truncated E polypeptides inducethe production of a titer of neutralizing antibodies against at leastone dengue virus of each of at least one, two, three, or four serotypesthat is about equal to or greater than a titer of neutralizingantibodies induced in the subject against the at least one dengue virusof each of at least one, two, three, or four serotypes, respectively, bya corresponding wild-type PRM15/truncated E polypeptide (“WTPRM15/truncated E fusion protein”) of the at least one dengue virus ofeach of the at least one, two, three or four serotypes, wherein eachsaid WT PRM15/truncated E protein polypeptide is selected from SEQ IDNOS:149-152. The sequence of SEQ ID NO:149, for example, comprises thefollowing amino acid sequence: a methionine as the first amino acidresidue, the last 15 amino acids from the C terminus of the prM sequenceof WT DEN-1, and a truncated amino acid sequence of DEN-1 envelopeprotein, which is termed “truncated” because it excludes a number ofamino acid residues from the C terminus of the envelope protein ofDEN-1. For example, in one embodiment, about 11-14% (and preferablyabout 13%) of the amino acid residues of the C terminus of the E proteinof Den-1 were excluded.

[0025] The invention also provides recombinant, synthetic, or mutantPRM15/truncated E polypeptides, each encoded by a nucleic acidcomprising a polynucleotide sequence selected from the group of: (a) apolynucleotide sequence having at least about 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or morenucleic acid sequence identity to at least one polynucleotide sequenceselected from the group of SEQ ID NOS:156-200, 235, 342, and 344, or acomplementary polynucleotide sequence thereof; (b) a RNA polynucleotidesequence comprising a DNA sequence selected from the group of SEQ IDNOS:156-200, 235, 342, and 344 in which all of the thymine nucleotideresidues in the DNA sequence are replaced with uracil nucleotideresidues or a complementary RNA polynucleotide sequence thereof; (c) aRNA polynucleotide sequence that has at least about 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore nucleic acid sequence identity to at least one RNA polynucleotidesequence of (b) or a complementary RNA polynucleotide sequence thereof;(d) a polynucleotide sequence that hybridizes under at least stringentconditions over substantially the entire length of a polynucleotidesequence of (a)-(c); (e) a polynucleotide sequence which would hybridizeunder at least stringent conditions over substantially the entire lengthof a polynucleotide sequence of any of (a)-(d) but for the degeneracy ofthe genetic code; and (f) a polynucleotide sequence that possesses anycombination of the features of the polynucleotide sequences of (a)-(e).

[0026] In another aspect, the invention provides recombinant, synthetic,mutant, and/or isolated polypeptides, each of which comprises an aminoacid sequence that has at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acidsequence identity to an amino acid sequence of at least one of SEQ IDNOS:139-148, 236-253, 343, and 345. Each such polypeptide typicallycomprises a recombinant, synthetic, or mutant dengue virus antigencomprising a fusion protein comprising a C15/full length prMprotein/full length E protein. C15/full length prM/full length E proteinpolypeptides induce an immune response in a subject against at least onedengue virus of each of at least one, two, three, or four dengue virusserotypes. Further, for some such polypeptides, the immune responseinduced against at least one dengue virus of each of at least 1, 2, 3 or4 serotypes is about equal to or greater than an immune response inducedin such cell against the at least one dengue virus of each of the atleast 1, 2, 3 or 4 serotypes by a wild-type C15/full length prM/fulllength E fusion protein polypeptide of each of the at least one, two,three or four serotypes, respectively, wherein the correspondingwild-type C15/full length prM/full length E fusion protein is selectedfrom SEQ ID NOS:227-230.

[0027] For C15/full length prM protein/full length E proteinpolypeptides, the immune response may comprise the production ofantibodies that bind to at least one dengue virus of each of at leastone, two, three or four serotypes. In addition, some such polypeptidesmay induce production of a number of antibodies that bind to at leastone dengue virus of each of the at least one, two, three, or fourserotypes that is about equal to or greater than that induced by acorresponding wild-type C15/full length prM/full length E fusion proteinof each of the at least one, two, three, or four serotypes,respectively, wherein each wild-type C15/full length prM/full length Efusion protein is selected from SEQ ID NOS:227-230.

[0028] In another aspect, some such C15/full length prM/full length Efusion protein polypeptides of the invention induce or produce a titerof neutralizing antibodies against at least one dengue virus of each ofat least one, two, three, or four dengue virus serotypes. Furthermore,some such polypeptides induce or produce a titer of neutralizingantibodies in a subject against at least one dengue virus of each of atleast one, two, three, or four serotypes that is about equal to orgreater than a titer of neutralizing antibodies induced or produced inthe subject against the at least one dengue virus of each of at leastone, two, three, or four serotypes by a wild-type C15/full lengthprM/full length E fusion protein polypeptide of the at least one denguevirus of each of the at least one, two, three, or four serotypes,respectively, wherein each said wild-type C15/full length prM/fulllength E fusion protein polypeptide is selected from SEQ ID NOS:227-230.

[0029] The invention also provides recombinant, synthetic, or mutantC15/full length prM/full length E fusion protein polypeptides, whereineach such polypeptide is encoded by a nucleic acid comprising apolynucleotide sequence selected from the group of: (a) a polynucleotidesequence having at least about 80%, 85%, 90%, 93%, 95%, 98% or morenucleic acid sequence identity to at least one polynucleotide sequenceselected from the group of SEQ ID NOS:201-210, 254-271, 342, and 344, ora complementary polynucleotide sequence thereof; (b) a RNApolynucleotide sequence comprising a DNA sequence selected from thegroup of SEQ ID NOS:201-210 254-271, 342, and 344 in which all of thethymine nucleotide residues in the DNA sequence are replaced with uracilnucleotide residues or a complementary RNA polynucleotide sequencethereof; (c) a RNA polynucleotide sequence that has at least about 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more nucleic acid sequence identity to at least one RNApolynucleotide sequence of (b) or a complementary RNA polynucleotidesequence thereof; (d) a polynucleotide sequence that hybridizes under atleast stringent conditions over substantially the entire length of apolynucleotide sequence of (a)-(c); (e) a polynucleotide sequence whichwould hybridize under at least stringent conditions over substantiallythe entire length of a polynucleotide sequence of any of (a)-(d) but forthe degeneracy of the genetic code; and (f) a polynucleotide sequencethat possesses any combination of the features of the polynucleotidesequences of (a)-(e).

[0030] In another aspect, the invention provides a compositioncomprising at least one recombinant, mutant, synthetic and/or isolatedpolypeptide or nucleic acid of the invention and an excipient orcarrier, including a pharmaceutically acceptable carrier or excipient.

[0031] In one aspect, the invention provides a composition comprising atleast one recombinant, mutant, synthetic and/or isolated polypeptidecomprising an amino acid sequence selected from the group of SEQ IDNOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and 345, or anantigenic or immunogenic polypeptide fragment thereof, that induces animmune response in a subject against at least one dengue virus of atleast one virus serotype that is about equal to or greater than theimmune response induced by a antigenic or immunogenic polypeptidefragment of the at least one dengue virus of the at least one serotype;and an excipient or carrier.

[0032] In yet another aspect, the invention provides an isolated,recombinant, mutant, or synthetic nucleic acid comprising apolynucleotide sequence selected from the group of: (a) a polynucleotidesequence having at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acidsequence identity to a sequence selected from the group of SEQ IDNOS:211-218 or a complementary polynucleotide sequence thereof; (b) aRNA polynucleotide sequence having at least about 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore nucleic acid sequence identity to a DNA sequence selected from thegroup of SEQ ID NOS:211-218 in which all of the thymine nucleotideresidues in the DNA sequence are replaced with uracil nucleotideresidues or a complementary RNA polynucleotide sequence thereof; and (c)a polynucleotide sequence that hybridizes under at least stringentconditions over substantially the entire length of a polynucleotidesequence of (a) or (b), or a complementary sequence thereof.

[0033] In another aspect, the invention includes an isolated,recombinant, mutant, or synthetic nucleic acid comprising apolynucleotide sequence selected from the group of: (a) a polynucleotidesequence comprising a nucleotide sequence having at least about 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more nucleic acid sequence identity to a sequenceselected from the group of SEQ ID NOS:285-330, or a complementarypolynucleotide sequence thereof; (b) a polynucleotide sequence encodinga polypeptide selected from SEQ ID NOS:1-49 and 153-155, or acomplementary polynucleotide sequence thereof; (c) a RNA polynucleotidesequence having at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to a DNA sequence selected from the group of SEQ ID NOS:285-330in which all of the thymine nucleotide residues in the DNA sequence arereplaced with uracil nucleotide residues, or a complementary RNApolynucleotide sequence thereof; (d) a polynucleotide sequence thathybridizes under at least stringent conditions over substantially theentire length of a polynucleotide sequence of (a)-(c); (e) apolynucleotide sequence which would hybridize under at least stringentconditions over substantially the entire length of a polynucleotidesequence of any of (a)-(d) but for the degeneracy of the genetic code;(f) a polynucleotide sequence having at least about 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to at least one polynucleotide sequence selectedfrom the group of SEQ ID NOS:285-330, or a complementary polynucleotidesequence thereof wherein said polynucleotide sequence encodes apolypeptide that induces an immune response in a subject against atleast one dengue virus of at least one serotype selected from dengue-1,dengue-2, dengue-3, and dengue-4 that is about equal to or greater thanan immune response induced in the subject against the at least onedengue virus of the at least one serotype by a wild-type truncatedenvelope (E) protein of at least one dengue virus of the at least oneserotype, wherein said wild-type truncated E protein is selected fromthe group of SEQ ID NOS:338-341; and (g) a polynucleotide sequence thatpossesses any combination of the features of the polynucleotidesequences of (a)-(f). The invention also provides a compositioncomprising an excipient or carrier and at least one nucleic acid of theinvention, including, e.g., at least one polynucleotide sequence asdefined by any of (a)-(g) above.

[0034] Also provided are isolated, recombinant, mutant, and/or syntheticnucleic acids that each comprise a polynucleotide sequence selected fromthe group of: (a) a polynucleotide sequence having at least about 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a sequence selected from thegroup of SEQ ID NOS:156-200, 235, 342, and 344, or a complementarypolynucleotide sequence thereof; (b) a polynucleotide sequence encodinga polypeptide selected from SEQ ID NOS:65-116, or a complementarypolynucleotide sequence thereof; (c) an RNA polynucleotide sequencehaving at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to aDNA sequence selected from the group of SEQ ID NOS:156-200, 235, 342,and 344,in which all of the thymine nucleotide residues in the DNAsequence are replaced with uracil nucleotide residues, or acomplementary RNA polynucleotide sequence thereof; (d) a polynucleotidesequence that hybridizes under at least stringent conditions oversubstantially the entire length of a polynucleotide sequence of (a)-(c);(e) a polynucleotide sequence which would hybridize under at leaststringent conditions over substantially the entire length of apolynucleotide sequence of any of (a)-(d) but for the degeneracy of thegenetic code; (f) a polynucleotide sequence or fragment thereof havingat least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at leastone polynucleotide sequence selected from the group of SEQ IDNOS:156-200, 235, 342, and 344, or a complementary polynucleotidesequence thereof, wherein said polynucleotide sequence or fragmentthereof encodes a polypeptide that induces an immune response in asubject against at least one dengue virus of at least one serotypeselected from DEN-1, DEN-2, DEN-3, and DEN-4 that is about equal to orgreater than an immune response induced in the subject against the atleast one dengue virus of the at least one serotype by a wild-typetruncated envelope (E) protein of at least one dengue virus of the atleast one serotype, wherein said wild-type truncated E protein isselected from any of SEQ ID NOS:149-152; and (g) a polynucleotidesequence that possesses any combination of features of the sequences of(a)-(f).

[0035] In another aspect, the invention provides isolated, recombinant,mutant, and/or synthetic nucleic acids that each comprise apolynucleotide sequence selected from the group of: (a) a polynucleotidesequence having at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to a sequence selected from the group of SEQ ID NOS:201-210,254-271, 342, and 344, or a complementary polynucleotide sequencethereof; (b) a polynucleotide sequence encoding a polypeptide selectedfrom SEQ ID NOS:139-148, 236-253, 343, and 345 or a complementarypolynucleotide sequence thereof; (c) a RNA polynucleotide sequencehaving at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to aDNA sequence selected from the group of SEQ ID NOS:201-210, 254-271,342, and 344 in which all of the thymine nucleotide residues in the DNAsequence are replaced with uracil nucleotide residues or a complementaryRNA polynucleotide sequence thereof; (d) a polynucleotide sequence thathybridizes under at least stringent conditions over substantially theentire length of a polynucleotide sequence of (a)-(c); (e) apolynucleotide sequence which would hybridize under at least stringentconditions over substantially the entire length of a polynucleotidesequence of any of (a)-(e) but for the degeneracy of the genetic code;(f) a polynucleotide sequence, or fragment thereof, having at leastabout 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to at least onepolynucleotide sequence selected from the group of SEQ ID NOS:201-210,254-271, 342, and 344, or a complementary polynucleotide sequencethereof, wherein said polynucleotide sequence or fragment thereofencodes a polypeptide that induces an immune response in a subjectagainst at least one dengue virus of at least one serotype selected fromDEN-1, DEN-2, DEN-3, and DEN-4 that is about equal to or greater than animmune response induced in the subject against the at least one denguevirus of the at least one serotype by a WT truncated envelope (E)protein of at least one dengue virus of the at least one serotype,wherein said WT truncated E protein is selected from the group of SEQ IDNOS:227-230; and (g) a polynucleotide sequence that possesses anycombination of the features of the polynucleotide sequences of (a)-(f).

[0036] Other advantageous features of the aforementioned polypeptidesand polynucleotides encoding such polypeptide include the ability toinduce a protective immune response against one or more dengue viruses,preferably against dengue viruses of multiple virus serotypes, in asubject, such as, e.g., an animal, including a mammal, or cell(s)thereof. Other desirable features of such polypeptides include higherexpression, higher secretion, and/or more or more specific antibodybinding exhibited by such polypeptides with respect to wild-type denguevirus proteins, including, e.g., C15/full length prM/full length Efusion proteins, full length prM/full length E fusion proteins,PRM15/truncated E protein polypeptides, PRM15/full length E fusionprotein, full length E or truncated E proteins, and/or one or morefragments of any thereof, as described herein.

[0037] The invention further provides fusion proteins comprising theaforementioned polynucleotides of the invention; vectors comprising oneor more of the polynucleotides of the invention; cells comprising suchpolypeptides, vectors, polynucleotides, and fusion proteins; andpharmaceutical compositions comprising such polypeptides,polynucleotides, vectors, fusion proteins, and/or cells. Exemplaryvectors provided by the invention include viral vectors, including,e.g., flaviviral vectors (including, e.g., attenuated flaviviral vectorscomprising a polynucleotide of the invention in place of at least aportion of the flaviviral vector genome encoding a wild-type C15/fulllength prM/full length E protein, full length prM/full length E fusionprotein, PRM15/truncated E protein polypeptide, PRM15/full length Efusion protein, full length E or truncated E protein, and/or fragmentsof any thereof) and nucleotide vectors, such as the plasmid vectorpMaxVax10.1 (described in Example 1 and shown in FIGS. 1 and 2), whichthe inventors have discovered to be an effective gene delivery vehiclein mammalian hosts.

[0038] With respect to polynucleotides, the invention further provides,for example, a polynucleotide comprising a nucleic acid sequence of atleast about 1200 nucleotides that has at least about 65%, 75%, 80%, 85%,or 90% nucleotide sequence identity with at least one of SEQ IDNOS:285-330, as well as polynucleotides which hybridize with such apolynucleotide, and polynucleotides comprising a sequence which is thecomplement of the nucleic acid sequence. In another aspect, theinvention provides a nucleic acid comprising a sequence of at leastabout 1200 nucleotides that has at least about 70%, 75%, 80%, or 90%nucleic sequence identity to at least one of SEQ ID NOS:211-214.

[0039] The invention also provides methods of promoting (inducing and/orenhancing) an immune response to a dengue virus in a subject (e.g.,animal, such as a mammal) by administering polypeptides, fusion protein,polynucleotides, vectors, or cells of the invention as described herein.In some such methods, administration of an immunogenic or antigenicpolypeptide of the invention (preferably, e.g., a polypeptide whichinduces a neutralizing antibody response against one or more dengueviruses of multiple virus serotypes) to a subject is followed by repeatadministration at selected time periods, resulting in an improved immuneresponse. Such “boosting” administration strategies also areadvantageously performed in conjunction with the administration of apolynucleotide of the invention (e.g., prophylactic or therapeuticadministration of an immunogen-encoding polynucleotide (e.g., DNAvaccine)) is preferably followed by subsequent administration ofadditional immunogen-encoding or antigen-encoding polynucleotide of theinvention and/or an immunogenic or antigenic polypeptide of theinvention).

[0040] In another aspect, the invention provides a compositioncomprising a library of at least two recombinant or synthetic nucleicacids obtained by a method comprising recombining at least a firstnucleic acid comprising a sequence selected from SEQ ID NOS:211-214, andat least a second nucleic acid, wherein the first and second nucleicacids differ from each other in two or more nucleotides, to produce alibrary of recombinant or synthetic nucleic acids.

[0041] The invention also includes a composition comprising a library ofnucleic acids obtained by a method comprising recombining at least afirst nucleic acid comprising a sequence selected from the group of SEQID NOS:215-218, and at least a second nucleic acid, wherein the firstand second nucleic acids differ from each other in two or morenucleotides, to produce a library of recombinant or synthetic nucleicacids.

[0042] In another aspect, the invention provides a polypeptide which isspecifically bound by polyclonal antisera raised against at least oneantigen, the at least one antigen comprising an amino acid sequenceselected from the group of SEQ ID NOS:1-49 and 153-155, or an antigenicor immunogenic fragment thereof, wherein said antigenic or immunogenicpolypeptide fragment thereof that induces an immune response in asubject against at least one dengue virus of at least one virus serotypethat is about equal to or greater than the immune response induced inthe subject by a antigenic or immunogenic polypeptide fragment of the atleast one dengue virus of the at least one serotype, wherein thepolyclonal antisera is subtracted with at least one of: a truncatedenvelope protein selected from the group of SEQ ID NOS:338-341 and atruncated envelope protein comprising a known wild-type truncated denguevirus protein sequence, or an amino acid sequence fragment of apolypeptide sequence corresponding to a known wild-type dengue virus Eprotein, wherein said amino acid sequence fragment has a lengthsubstantially identical (e.g., at least about 75%, 80%, 85%, 86%, 87%,88% or 89%, preferably at least about 90%, 91%, 92%, 93%, or 94%, andmore preferably at least about 95% (e.g., about 87-95%), 96% 97%, 98%,99%, 99.5% sequence identity) to the truncated envelope protein of anyof SEQ ID NOS:338-341.

[0043] The invention also provides polypeptides that are specificallybound by polyclonal antisera raised against at least one antigen, the atleast one antigen comprising an amino acid sequence selected from thegroup of SEQ ID NOS:65-116, wherein the polyclonal antisera issubtracted with at least one of: a PRM15/truncated envelope proteinselected from the group of SEQ ID NOS:149-152 and other amino acidsequences comprising known dengue virus PRM15/truncated envelopeproteins.

[0044] The invention also provides polypeptides that are specificallybound by polyclonal antisera raised against at least one antigen, the atleast one antigen comprising an amino acid sequence selected from thegroup of SEQ ID NOS:139-148, 236-253, 343, and 345, wherein thepolyclonal antisera is subtracted with at least one of: a fusion proteincomprising a C15/full length prM protein/full length E protein selectedfrom the group of SEQ ID NOS:227-230 and other amino acid sequencescomprising known dengue virus C15/full length prM protein/full length Eprotein PRM15/truncated envelope proteins.

[0045] The invention also includes an antibody or antisera produced byadministering a truncated E polypeptide of the invention to a subject,which antibody or antisera specifically binds at least one antigen, theat least one antigen comprising a polypeptide comprising at least oneamino acid sequence of SEQ ID NOS:1-49 and 153-155, which antibody orantisera does not specifically bind to at one or more of: thepolypeptides of SEQ ID NOS:338-341 and a truncated envelope proteincomprising a known wild-type truncated dengue virus protein sequence, oran amino acid sequence fragment of a polypeptide sequence correspondingto a known wild-type dengue virus E protein, wherein said amino acidsequence fragment has a length substantially identical (e.g., at leastabout 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at least about90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity) to thetruncated envelope protein of any of SEQ ID NOS:338-341.

[0046] In another aspect, the invention provides an antibody or antiseraproduced by administering a truncated E polypeptide of the invention toa subject, which antibody or antisera specifically binds at least oneantigen, the at least one antigen comprising a polypeptide comprising atleast one amino acid sequence of SEQ ID NOS:65-116, which antibody orantisera does not specifically bind to at one or more of: aPRM15/truncated envelope protein selected from the group of SEQ IDNOS:149-152 and other amino acid sequences comprising known dengue virusPRM15/truncated envelope proteins. In yet another aspect, the inventionprovides an antibody or antisera produced by administering a truncated Epolypeptide of the invention to a subject, which antibody or antiseraspecifically binds at least one antigen, the at least one antigencomprising a polypeptide comprising at least sequence of SEQ IDNOS:139-148, 235-253, 343, and 345, which antibody or antisera does notspecifically bind to at one or more of: a fusion protein comprising aC15/full length prM protein/full length E protein selected from thegroup of SEQ ID NOS:227-230 and other amino acid sequences comprisingknown dengue virus C15/full length prM protein/full length E proteinPRM15/truncated envelope proteins.

[0047] The invention also includes a pharmaceutical compositioncomprising at least one polypeptide of the invention (or at least onepolynucleotide of the invention), and a pharmaceutically acceptablediluent, carrier, or excipient, wherein the at least one polypeptide (orpolynucleotide) is present in an amount effective to provide a subjectwith protective immunity to at least one dengue virus of at least 1, 2,3, or 4 dengue serotypes.

[0048] The invention also includes a pharmaceutical compositioncomprising at least one polypeptide of the invention (or at least onepolynucleotide of the invention), and a pharmaceutically acceptablediluent, carrier, or excipient, wherein the at least one polypeptide (orpolynucleotide) is present in an amount effective to induce an immuneresponse (e.g., specific immune response) to at least one dengue virusof at least one, two, three, or four dengue virus serotypes and/orprovide a subject with protective immunity to at least one dengue virusof at least one, two, three, or four dengue virus serotypes.

[0049] In another aspect, the invention provides a vaccine comprising atleast one polypeptide of the invention (or polynucleotide of theinvention) in an amount effective to provide a subject with protectiveimmunity to at least one dengue virus of at least one, two, three, orfour virus serotypes, and a pharmaceutically acceptable diluent,carrier, or excipient.

[0050] Also provided are methods of producing antibodies to at least onedengue virus of at least one serotype in a subject which compriseadministering to said subject at least one nucleic acid or polypeptideof the invention, or a combination of both. Also included are methods ofproducing one or more antibodies that bind to at least one dengue virusof at least one serotype which comprise administering an effectiveamount of a polypeptide and/or nucleic acid of the invention, or acomposition of either or both, to a population of cells such that thecells produce one or more antibodies that bind to at least one denguevirus of at least one serotype.

[0051] The invention further provides a method of producing a protectiveimmune response against at least one dengue virus of each of at leastone, two, three or four dengue virus serotypes in a subject, wherein themethod comprises administering to the subject an amount effective of atleast one nucleic acid of the invention sufficient to produce aprotective immune response against challenge by the at least one denguevirus of each of the at least one, two, three, or four serotypes,respectively. For some such methods, the immune response is a protectiveantibody response, such that when said at least one nucleic acid isexpressed, antibodies to at least one dengue virus of each of at leastone, two, three, or four serotypes are generated in the subject at alevel sufficient to produce a protective antibody response againstchallenge by the at least one dengue virus of each of the at least one,two, three of four serotypes, respectively.

[0052] In another aspect, the invention provides a method of inducing animmune response in a subject, such as a mammal, to at least one denguevirus of at least one serotype comprising administering an effectiveamount of at least one polypeptide of the invention, or an effectiveamount of a composition thereof, or both, to a subject. The inventionalso provides a method of inducing an immune response in a subject to atleast one dengue virus of at least one serotype comprising administeringan effective amount of at least one nucleic acid of the invention, or aneffective amount of a composition thereof, or both, to a subject. Theeffective amount is usually an immunogenic or antigenic amount thatfacilitates induces a protective immune response or therapeutic orprophylactic treatment.

[0053] In addition, the invention provides a method of promoting animmune response in a subject to at least one dengue virus of at leastone serotype comprising introducing at least one nucleic acid orpolypeptide of the invention into a population of cells and delivering(e.g., implanting) the cells in a subject. The population of cells mayinitially have been obtained from the subject before introduction of thenucleic acid or polypeptide.

[0054] In another aspect, the invention provides a target nucleic acidwhich, but for the degeneracy of the genetic code, hybridizes under atleast stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from: SEQ IDNOS:65-116, wherein the unique subsequence is unique as compared to aknown dengue virus antigen envelope polypeptide, any polypeptideselected from SEQ ID NOS:149-150, or a polypeptide encoded by any of SEQID NOS:231-234.

[0055] In another aspect, the invention provides a recombinant orsynthetic polypeptide comprising an amino acid sequence that has atleast about 90% amino acid sequence identity to an amino acid sequencecomprising a polypeptide fragment of at least one polypeptide sequenceselected from SEQ ID NOS:236-253, wherein the polypeptide fragment doesnot include the first 16 amino acid residues of the selected polypeptidesequence of SEQ IDS:236-253, and wherein the recombinant or syntheticpolypeptide induces an immune response in a subject or cells of thesubject against at least one dengue virus of each of at least twoserotypes selected from the group of dengue-1, dengue-2, dengue-3, anddengue-4 that is about equal to or greater than an immune responseinduced in the subject or cells thereof against the at least one denguevirus of the at least two serotypes by a wild-type envelope (E) proteinof at least one dengue virus of each of the at least two serotypesselected from SEQ ID NO:338, 339, 340, and 341, respectively.

[0056] Also included is a recombinant or synthetic polypeptidecomprising an amino acid sequence that has at least about 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more amino acid sequence identity to an amino acid sequence of atleast one of SEQ ID NOS:1-49 and 153-155, wherein the recombinant orsynthetic polypeptide induces an immune response in a subject or apopulation of cells thereof against at least one dengue virus of each ofat least two serotypes selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4 that is about equal to or greater than thatinduced by a WT E protein of each of said at least one dengue virus,respectively, wherein said WT E protein has an amino acid sequencelength substantially equivalent or identical to that of the recombinantor synthetic polypeptide.

[0057] In another aspect, the invention provides a recombinant orsynthetic polypeptide comprising an amino acid sequence that has atleast about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to anamino acid sequence of at least one of SEQ ID NOS:1-49 and 153-155,wherein the recombinant or synthetic polypeptide induces an immuneresponse in a subject, e.g., mammal, or cells thereof, against at leastone dengue virus of each of at least two serotypes selected from thegroup of dengue-1, dengue-2, dengue-3, and dengue-4 that is about equalto or greater than an immune response induced in the subject, or cellsthereof, against each said at least one dengue virus of each of the atleast two dengue serotypes induced by a WT truncated E protein of eachof dengue-1, dengue-2, dengue-3, and dengue-4, respectively, whereinsaid WT truncated E protein has an amino acid sequence lengthsubstantially equivalent or identical to that of the recombinant orsynthetic polypeptide.

[0058] In yet another aspect, the invention provides a recombinant orsynthetic polypeptide comprising an amino acid sequence that has atleast about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to theamino acid sequence of at least one of SEQ ID NOS:65-116, wherein therecombinant or synthetic polypeptide induces an immune response in asubject or population of cells thereof, e.g., mammal or population ofmammalian cells, against at least one dengue virus of each of at leasttwo serotypes selected from the group of DEN-1, DEN-2, DEN-3, and DEN-4that is about equal to or greater than an immune response induced in thesubject (e.g., mammal or population of mammalian cells) against eachsaid at least one dengue virus of each of the at least two serotypes bya WT PRM15/truncated E protein of each of DEN-1, DEN-2, DEN-3, andDEN-4, respectively, wherein each said WT PRM15/truncated E proteinpolypeptide is selected from SEQ ID NOS:149-152.

[0059] The invention further provides a recombinant or syntheticpolypeptide comprising an amino acid sequence that has at least about70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more amino acid sequence identity to an amino acidsequence of at least one of SEQ ID NOS:139-148, 236-253, 343, and 345,wherein the recombinant or synthetic polypeptide induces an immuneresponse in a subject or population of cells thereof against at leastone dengue virus of each of at least two serotypes selected from thegroup of DEN-1, -2, -3, and -4 virus that is about equal to or greaterthan an immune response induced in the mammalian cell against the atleast one dengue virus of each of the at least two serotypes by a WTC15/full length prM/full length E fusion protein of each of said atleast two serotypes, wherein each said WT C15/full length prM/fulllength E fusion protein is selected from SEQ ID NOS:227-230.

[0060] Also included is a protein aggregate formed from a population ofat least two recombinant or synthetic polypeptides of the invention.Such protein aggregate includes dimers, trimers, etc. and othermultiples of the polypeptides of the invention, which polypeptides neednot be identical.

[0061] Included are virus-like particles comprising of at least twopolypeptides of the invention, which polypeptides need not be identical.Some such virus-like particles are formed from a population of at leasttwo polypeptides selected from among the recombinant, mutant, orsynthetic polypeptides. Some such virus-like particles are formed fromexpression of one or more nucleic acids encoding at least twopolypeptides of the invention.

[0062] Also provided are viruses, including, e.g., attenuated viruses,comprising at least one of: (1) a nucleic acid of the invention; (2) apolypeptide of the invention; and/or (3) a vector of the invention. Thevirus may comprise a yellow fever (YF) virus that has been modified witha nucleic acid, polypeptide, and/or vector of the invention. Included isa chimeric virus comprising a dengue virus (e.g., DEN-2 or DEN-4) thatcomprises at least one polypeptide of the invention in place of or inaddition to the respective dengue virus full length or truncated Eprotein, the respective dengue virus full length prM protein or fragmentthereof (e.g., PRM15), and/or the respective dengue virus fusion proteincomprising the native dengue virus C15/full length prM/full lengthenvelope protein. Included is an attenuated or replication-deficientchimeric flavivirus (e.g., dengue virus or YF virus) or adenovirus,comprising at least one nucleic acid or polypeptide of the invention inplace of the corresponding nucleic acid or polypeptide of the flavivirusor adenovirus, respectively.

[0063] Also provided is a DNA or RNA construct or a viable chimericrecombinant flavivirus, said DNA or RNA construct or chimericrecombinant flavivirus comprising a first region of nucleic acidencoding a recombinant protein(s) (e.g., PRM15/truncE, C15/full prM/fullE, or prM & E proteins) of the invention) operably linked to a 2ndregion of nucleic acid encoding non-structural proteins of a flavivirus(e.g., YF virus or DEN-2, DEN-4).

[0064] The invention also includes an integrated system comprising acomputer or computer readable medium comprising a database comprising atleast one sequence record, each said at least one sequence recordcomprising at least one character string corresponding to at least onepolypeptide sequence selected from the group of SEQ ID NOS:1-49, 65-116,139-148, 153-155, 236-253, 343, and 345, or at least nucleic acidsequence selected from the group of SEQ ID NOS:156-210, 235, 254-271,285-330, 342, and 344, the integrated system further comprising a userinput interface allowing a user to selectively view said at least onesequence record.

[0065] Also provided is a method of using a computer system to presentinformation pertaining to at least one of a plurality of sequencerecords stored in a database, the sequence records each comprising atleast one character string corresponding to SEQ ID NOS:1-49, 65-116,139-148, 153-210, 235-271, 285-330, 342-345, the method comprising: (a)determining a list of at least one character string corresponding to atleast one of the group of SEQ ID NOS:1-49, 65-116, 139-148, 153-210,235-271, 285-330, 342-345, or a subsequence thereof; (b) determiningwhich said at least one character string of the list is selected by auser; and (c) displaying each selected character string, or aligningeach selected character string with an additional character string.

[0066] The invention further provides improved and novel methods andtechniques for detecting and/or diagnosing the presence of antibodiesagainst one, two, three or four dengue virus serotypes in a sample(including, e.g., a biological sample, such as a serum sample obtainedfrom a subject, such as a animal, including, e.g., a mammal, including,e.g., a human at risk for dengue virus infection). The invention alsoprovides methods and techniques for simultaneously detecting and/ordiagnosing the presence of antibodies against one, two, three or fourdengue virus serotypes in a single sample. The polypeptides of theinvention, and the nucleic acids encoding them, can be used in thisrespect to detect or diagnose a biological sample for the presence ofsuch antibodies. Advantageously, a composition comprising as little asabout 10 μl of the aspirated supernatant of a cell culture transfectedwith a polynucleotide of the invention can serve as a uniform andsuitable substrate for the simultaneous diagnosis or detection ofantibodies against all four serotypes of dengue virus in a sample, suchas, e.g., a biological sample, including a sample obtained from asubject, such as a mammal, including, e.g., human, at risk for denguevirus infection. The biological sample can be, e.g., a blood serumsample obtained from a subject.

[0067] In another aspect, the invention provides a method of diagnosinga sample for, or detecting in a sample, the presence of one or moreantibodies that bind to at least one dengue virus of at least oneserotype, the method comprising: (a) contacting the sample with at leastone polypeptide of the invention under conditions such that if thesample comprises one or more antibodies that bind to the at one denguevirus, at least one anti-dengue virus antibody binds to the at least onepolypeptide to form a mixed composition; (b) contacting the mixedcomposition with at least one affinity molecule that binds to ananti-dengue virus antibody; (c) removing unbound affinity-molecule fromthe mixed composition; and (d) diagnosing or detecting the presence orabsence of one or more affinity molecules, wherein the presence of oneor more affinity molecules is indicative of the presence of one or moreantibodies that bind to the at least one dengue virus in the sample.

[0068] In addition, the invention provides a method of diagnosing asample for, or detecting in a sample, the presence of at least oneantibody that binds to a dengue virus of at least one serotype, saidmethod comprising: (a) contacting the sample with at least onepolypeptide of the invention under conditions such that if the samplecomprises one or more antibodies that bind to the dengue virus, at leastone anti-dengue virus antibody binds to the at least one polypeptide toform at least one antibody-polypeptide complex, and (b) diagnosing ordetecting the presence or absence of the at least oneantibody-polypeptide complex, wherein the presence of the at least oneantibody-polypeptide complex is indicative of the presence of at leastone antibody that binds to the dengue virus in the sample.

[0069] Methods of producing the above-described nucleic acids andpolypeptides also are provided by the present invention. In general, thepolynucleotides of the invention are advantageously prepared by standardnucleic acid synthesis techniques (e.g., polymerase chain reaction(PCR)-facilitated overlapping nucleic acid assembly). However, theinvention also provides a method of performing recursive sequencerecombination with selected nucleic acids of the invention andappropriate screening techniques to generate and identify novel dengueantigen-encoding nucleic acids. Methods of making polypeptides of theinvention include the transfection or infection of cells, either invitro, in vivo, or ex vivo, with the polynucleotides and/or vectors ofthe invention, in addition to standard synthetic protein synthesistechniques.

[0070] Numerous alternative, additional and/or more particularpolynucleotides, polypeptides, fusion proteins, viruses, vectors, cells,cell cultures, compositions, transgenic animals, transdermal patches,methods of using nucleic acids and/or polypeptides of the invention,diagnostic and detection assays, methods and techniques provided by theinvention are set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0071]FIG. 1 is a map of a representative pMaxVax10.1 vector.

[0072]FIG. 2 illustrates an exemplary pMaxVax10.1 vector comprising aninsertion of a nucleic acid that encodes a fusion protein comprising aPRM15 signal peptide/truncated E protein of a wild-type DEN-2 virus. ApMaxVax10.1 vector may be similarly constructed with a nucleic acid thatencodes a fusion protein comprising a PRM15 signal peptide/truncated Eprotein of a WT DEN-1, DEN-3, or DEN-4 virus, or a variant thereof, insubstituted for the nucleic acid sequence encoding the PRM15 signalpeptide/truncated E protein of WT DEN-2 (as shown in FIG. 2). Thetruncated E protein of DEN-2 polyprotein comprised a polypeptidesequence comprising about 90% of the contiguous amino acid residues ofthe sequence of the DEN-2 E protein as measured in sequence order fromthe N terminus of the E protein, as explained in detail below. The term“truncated E protein” is abbreviated as “tE” or “tE protein.” Theinvention also provides a pMaxVax10.1 vector comprising a nucleic acidof the invention that encodes a recombinant or synthetic dengue virusantigen of the invention; such nucleic acid is similarly substitutedinto the pMaxVax10.1 vector in the same position as the nucleic acidencoding the DEN-2 PRM15/truncated E protein polypeptide shown in FIG.2.

[0073]FIG. 3 displays a Western blot of dengue virus antigens, eachcomprising a PRM15 signal peptide/truncated (t) E protein, expressed inthe lysate (L) and secreted into the medium supernatant (SN) from 293cells transfected with one of the following vectors:pMaxVax10.1_(Den-1PRM15/tE CO), pMaxVax10.1_(Den-2PRM15/tE CO),pMaxVax10.1_(Den-3PRM15/tE CO), and pMaxVax10.1_(Den-4PRM15/tE CO). Thenitrocellulose membrane bound proteins were incubated with antibodiesfrom mouse ascitic fluid for DEN-1, DEN-2, DEN-3, and DEN-4.

[0074]FIG. 4 includes a series of dot blots obtained by FACS analysis of293 cells transfected with either pMaxVax10.1_(2/7) vector orpMaxVax10.1_(Den-3PRM15/tE CO) vector and separately incubated withmouse anti-DEN-1, DEN-2, DEN-3, or DEN-4 antisera.

[0075]FIG. 5 is a Western blot of recombinant PRM15/truncated E denguevirus antigens of the invention expressed from a pMaxVax10.1 DNA vector,compared to PRM15/truncated E antigens having wild-type (wt) amino acidsequences expressed from pMaxVax10.1_(Den-1PRM15/tE CO),pMaxVax10.1_(Den-2PRM15/tE CO), pMaxVax10.1_(Den-3PRM15/tE CO), andpMaxVax10.1_(Den-4PRM15/tE CO) vectors, bound with DEN-1, DEN-3, andDEN-4 murine antisera and appropriate secondary antibodies. Nucleotidesequences encoding these recombinant PRM15/tE dengue virus antigens werescreened or selected from among recombinant nucleotide sequences inrecombinant libraries produced by recursive sequence recombination. Inthe figure, the letter “C” refers to a control vector and represents thedata obtained by transfecting 293 cells with pMaxVax10.1_(null) vectorthat does not include a dengue virus nucleotide sequence.

[0076]FIG. 6 shows the optical density (OD) values obtained by ELISAanalysis of antisera obtained from mice, each of which had been injectedwith a pMaxVax10.1 vector comprising a representative recombinant (e.g.,shuffled) nucleic acid sequence identified in libraries of recombinantnucleic acids using ELISA plates, coated with inactivated DEN-1, DEN-2,DEN-3, and DEN-4 viruses. The term “WT” refers to a pMaxVax10.1 vectorcomprising a nucleic acid sequence encoding PRM15/truncated envelopeprotein of a wild-type DEN-1, DEN-2, DEN-3, or DEN-4 polyprotein,respectively. The control vector comprises a pMaxVax10.1_(null) vector(termed “pMV”) lacking any dengue virus nucleic acid sequence.

[0077]FIG. 7 presents a comparison of ELISA OD values for antiseraobtained from blood of mice injected with a pMaxVax10.1 vectorcomprising a parental dengue virus PRM15/tE nucleic acid (encoding a WTDEN-1 DEN-2, DEN-3, or DEN-4 polypeptide sequence) or a selectrepresentative recombinant PRM15/tE nucleic acid encoding a recombinantPRM15/tE dengue virus antigen (Ag). Results using antisera obtained frommice injected with a mixture of four pMaxVax10.1 vectors, each vectorincluding a nucleotide sequence encoding one of the four WT DEN-1PRM15/tE, DEN-2PRM15/tE, DEN-3PRM15/tE, and DEN-4PRM15/tE antigens(i.e., pMaxVax10.1_(DEN-3PRM15/tE CO), pMaxVax10.1_(DEN-2PRM15/tE CO),pMaxVax10.1_(DEN-3PRM15/tE CO), and pMaxVax10.1_(DEN-41PRM15/tE CO)),are also shown. The control (“pMV”) is a pMaxVax10.1 vector that lacks aparental or recombinant PRM15/tE nucleic acid.

[0078]FIG. 8A illustrates the results of reciprocal 50% plaque reductionneutralization titers (PRNT) for sera obtained from mice at day 76 afterthe initial injection at day 0 with a representative pMaxVax10.1 DNAplasmid vector comprising either one of the four WT DEN-1PRM15/tE,DEN-2PRM15/tE, DEN-3PRM15/tE, and DEN-4PRM15/tE antigens, or a mix ofthese 4 wild-type antigens, or a recombinant nucleotide sequence of theinvention corresponding to one of the following antigens: 18E9, 18D7,16G11, 18H2, 16B4,6E12, 2G11, 2/7, 15D4, and 18H6. FIG. 8B illustratesthe results of reciprocal 50% plaque reduction neutralization titers(PRNT) for sera obtained from mice at day 76 after the initial injectionat day 0 with a representative pMaxVax10.1 DNA plasmid vector comprisingeither one of the four WT DEN-1, DEN-2, DEN-3, and DEN-4 antigens in theC15/full prM/full E antigen format, or a mix of these 4 wild-typeantigens, or a recombinant nucleotide sequence of the inventioncorresponding to one of the following C15/full prM/full E antigens:5/21-D1, 2G11-D4, and 6E12-D4.

[0079]FIG. 9 shows a Western blot analysis of nine secreted recombinantdengue virus polypeptide antigens of the invention, each encoded by arecombinant polynucleotide of the invention, that induced a neutralizingantibody response against wild-type dengue viruses of at least twodengue virus serotypes in vivo. “E” refers to envelope protein, and thearrow to the right of the blot indicates the position of the E proteinin the blot. For each recombinant antigen, the number of differentserotypes neutralized by the antibody response induced by the antigen isshown. The PRNT results are shown in FIGS. 8A and 8B.

[0080]FIG. 10 provides OD graphs obtained by ELISA analyses of antiseraobtained from mice, each of which had been injected with a pMaxVax10.1vector comprising a recombinant nucleotide sequence. The recombinantnucleotide sequences were generated by recursive sequence recombinationof the human codon optimized dengue virus sequences (e.g., SEQ IDNOS:215-218). Each such recombinant nucleotide sequence encoded arecombinant dengue virus antigen having the following format: C15 signalsequence/full length prM protein/full length E protein. ApMaxVax10.1_(null) vector (“pMV”) served as the control vector.

[0081]FIG. 11 shows the results of a sequence diversity analysis (e.g.,chimerism) of amino acid sequences of representative recombinantPRM15/truncated E dengue virus antigens of the invention that areencoded by recombinant nucleic acids of the invention, as compared withsequences of the parental WT DEN-1 PRM15/tE, DEN-2 PRM15/tE, DEN-3PRM15/tE, and DEN-4 PRM15/tE proteins. This analysis confirms that theserecombinant antigens include amino acid fragments or segments from allfour parental WT protein sequences and thus constitute chimeras of theparental sequences.

[0082]FIG. 12 is a graph showing the percentage of mice that havesurvived challenge with DEN-2 virus over a period of 28 days followingimmunization with a recombinant pMaxVax10.1 vector of the invention. Inthis experiment, a recombinant pMaxVax10.1 vector comprising one of thefollowing three recombinant PRM15/tE nucleic acid sequences of theinvention and a mixture of these three nucleic acid sequences wereevaluated: SEQ ID NOS:235 (18H6), SEQ ID NOS:204 (2G11-D4) and SEQ IDNOS: 202 (6E12-D4). For comparison, some mice were injected with apMaxVax10.1 vector comprising a WT DEN-2 PRM15/tE nucleic acid sequenceand WT DEN-2 C15/full prM/full E, or a WT DEN-3 C15/full prM/full Enucleic acid sequence, as well as a mix of all four DEN-1-4 WT PRM15/tEand C15/full prM/full E. Mice injected with PBS or a pMaxVax10.1_(null)vector served as control mice.

[0083]FIGS. 13A and 13B show dot blots of 10 μl to 100 μl of supernatantfrom 293 cells transfected with representative nucleic acids of theinvention (e.g., 5/21 (SEQ ID NO:157), 2/7 (SEQ ID NO:156), 6E12 (SEQ IDNO:159) and 2G11 (SEQ ID NO:157) when reacted with mouse DEN-1, DEN-2,DEN-3, and DEN-4 antisera.

[0084]FIG. 14 is an exemplary graphical representation of a wild-typedengue virus gene and dengue virus structural proteins encoded therefrom(capsid, prM, and E proteins).

[0085]FIGS. 15A and 15B show DEN-3 WT in C15/full prM/full E proteinformat and selected recombinant polypeptides of the invention (18H6 inPRM15/truncated E format and 16G11-25B10 in C15/full prM/full E format)purified by centrifugation through 20%-60% sucrose gradients. Thepurified polypeptides are either stained with Coomassie Blue (15A) in apolyacrylamide (PAA) gel or with DEN-specific antibodies after WesternBlot transfer to nitrocellulose filters (15B) using standard techniques(see, e.g., Rapley, R. and Walker, J. M. eds., MOLECULAR BIOMETHODSHANDBOOK (1998), Humana Press, Inc., Tijssen (1993) LABORATORYTECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY—HYBRIDIZATION WITHNUCLEIC ACID PROBES [hereinafter Rapley and Walker, MOLECULAR BIOMETHODSHANDBOOK]. E represents “envelope” protein. Bovine serum albumin (BSA)was used as a control in the gel and Western Blot. Bands suggestingformation of a dimer polypeptide for each of DEN-3 C15/full prM/full Eand 16G11-25B10 are shown.

[0086]FIG. 16 presents a comparison of ELISA OD values for antiseraobtained from blood of mice immunized by injection with either apMaxVax10.1 vector encoding one of the following recombinant polypeptideantigens of the invention (18H6 (SEQ ID NO:235) or 16G11-25B10 (SEQ IDNO:255)) or by sucrose gradient purified polypeptides of the invention(18H6 (SEQ ID NO:110) or 16G11-25B10 (SEQ ID NO:251)) in alum adjuvant.The mice received 2 booster immunizations in 3-week intervals, eitherwith a pMaxVax10.1 vector encoding one of the following recombinantpolypeptide antigens of the invention (e.g., DNA+DNA), or withpolypeptides in alum after initial injection with DNA (e.g.,DNA+Protein) or protein (e.g., Protein+Protein). “Prot.” represents“protein.”

[0087]FIG. 17 presents a comparison of ELISA optical density values forantisera obtained at different time points (days 0, 28, 56, and 112)from blood of 6 monkeys per group, individually injected at days 0, 28,and 84 with: (1) a mixture comprising four pMaxVax10.1 nucleic acidvectors in PBS, each vector comprising one of four parental WT denguevirus C15/full length prM/full length E nucleic acids (4WT,Mix) encodinga WT DEN-1 DEN-2, DEN-3, or DEN-4 C15/full length prM/full length Epolypeptide, respectively; (2) a pMaxVax10.1 vector comprising arepresentative recombinant PRM15/tE nuclei acid (18H6) that encodes arecombinant PRM15/tE dengue virus antigen; (3) a pMaxVax10.1 vectorcomprising a representative PRM C15/full length prM/full length Enucleic acid (6E12-D4 and 2G11-D4), each of which encodes a C15/fulllength prM/full length E dengue virus antigen; or (4) a mixture of threepMaxVax10.1 vectors in PBS, each vector comprising a nucleic acidcorresponding to one of 18H6, 6E12-D4, and 2G11-D4 (3 Sh,Mix). Thecontrol (“pMV ctrl”) is a pMaxVax10.1 vector that lacks a parental orrecombinant PRM15/tE or C15/full length prM/full length E nucleic acid.All nucleic acid vectors were administered as compositions in PBS.

DETAILED DESCRIPTION OF THE INVENTION

[0088] The invention provides novel recombinant, synthetic, mutant,and/or isolated polypeptides, fusion proteins, antibodies, nucleic acids(e.g., polynucleotides), viruses, virus-like particles; vectorscomprising such nucleic acids and/or encoding such polypeptides, fusionproteins, antibodies, viruses, and virus-like particles; cells andcompositions comprising such nucleic acids, polypeptides, fusionproteins, antibodies, viruses, virus-like particles and/or vectors; andvaccines comprising one or more of the aforementioned nucleic acids,polypeptides, fusion proteins, antibodies, viruses, virus-likeparticles, vectors, cells, and compositions of the invention. Theinvention further provides methods of making and methods of using suchnucleic acids, polypeptides, fusion proteins, antibodies, viruses,virus-like particles, vectors, cells, and compositions of the invention.

[0089] In one aspect, the nucleic acids, polypeptides, fusion proteins,vectors, viruses, virus-like particles, cells, antibodies andcompositions are generally useful in modulating, promoting, inducing,and/or enhancing an immune response(s) to one or more flavivirusesand/or one or more dengue viruses of one or more serotypes, including,but not limited to, e.g., dengue-1, dengue-2, dengue-3, and dengue-4, orvariants thereof, and/or analyzing biological samples for the presenceof anti-flavivirus antibodies against at least one flavivirus of atleast one flavivirus serotype or variant thereof, and more particularlyanti-dengue virus antibodies against at least one of DEN-1 DEN-2, DEN-3,and DEN-4 viruses or a variant thereof. The nucleic acids, polypeptides,fusion proteins, vectors, viruses, virus-like particles, antibodies,cells, compositions, and methods of the invention described herein arealso believed useful in in vivo methods for the prophylactic and/ortherapeutic treatment of animals (including, e.g., vertebrates andmammals) of a disease(s) associated with at least one flavivirus of atleast one serotype or a variant thereof (including, e.g., at least onedengue virus of at least one serotype or variant thereof), and inmethods for the in vitro, ex vivo, and/or in vivo diagnosis, detection,and/or identification at least one flavivirus or variant thereof(including, e.g., at least one dengue virus of at least one serotype orvariant thereof).

[0090] In one aspect, the nucleic acids, polypeptides, fusion proteins,vectors, viruses, virus-like particles, antibodies, cells, compositions,and methods of the invention are particularly useful in in vivo, and/orex vivo methods of inducing or enhancing an immune response in an animalto at least one virus of the Flaviviridae family of viruses (e.g., amember of the Flavivirus genus such as Japanese encephalitis virus) andmethods for the prophylactic and/or therapeutic treatment of animals(including, e.g., vertebrates and mammals) of a disease(s) associatedwith at least one virus that is a member of the Flaviviridae family ofviruses, which includes flaviviruses, pestiviruses, and hepaciviruses,or variant of any such virus thereof, preferably wherein said at leastone virus or variant thereof is a virus that is related to at least onedengue virus of at least one serotype (including, e.g., but not limitedto, a virus of the Flaviviridae family, such as a member of theFlavivirus genus or another flavivirus, including, e.g., a yellow fevervirus, St. Louis encephalitis virus, Japanese encephalitis virus,ticke-borne encephalitis virus, Murray Valley encephalitis virus,Russian spring-summer encephalitis virus, and/or West Nile virus, andincluding, e.g., those viruses that are described as being related todengue viruses in FIELDS VIROLOGY, supra, Vol. 1, Chapters 32 and 33(4^(th) ed.), or any variant of any such virus).

[0091] In another aspect, the nucleic acids, polypeptides, fusionproteins, vectors, viruses, virus-like particles, antibodies, cells,compositions, and methods of the invention are useful in methods for thein vitro, ex vivo, and/or in vivo diagnosis, detection, and/oridentification at least one virus of the Flaviviridae family (e.g.,which consists of three genera, flavivirus, pestivirus, and hepacivirus)or a variant of any such virus thereof, preferably wherein said at leastone virus or variant thereof is a virus that is related to at least onedengue virus (including, e.g., but not limited to, a virus of theFlaviviridae family, another flavivirus, yellow fever viruses, St. Louisencephalitis viruses, Japanese encephalitis viruses, ticke-borneencephalitis viruses, Murray Valley encephalitis virus, Russianspring-summer encephalitis virus, and/or West Nile viruses, andincluding, e.g., those viruses that are described as being related todengue viruses in FIELDS VIROLOGY, supra, Vol. 1, Chapters 32 and 33(4^(th) ed.), or any variant of any such virus). As such, it will beunderstood that a reference to a dengue virus in the following detaileddescription can be construed as relating more generally to a virus ofthe Flaviviridae virus family, including a flavivirus, pestivirus, orhepacivirus, particularly to member of the Flavivirus genus, preferablyto a flavivirus of at least one serotype, and especially to a flavivirusthat is related or, preferably, closely related to at least one denguevirus of at least one serotype, unless otherwise stated or clearlycontradicted by context. It will be understood that a reference to apolypeptide in the following detailed description can be construed,depending upon the context, as including a fusion protein.

[0092] The polypeptides, fusion proteins, nucleic acids, vectors,viruses, virus-like particles, compositions, cells, and methods of theinvention are also useful in in vivo and/or ex vivo methods of inducingan immune response in animals and/or in in vivo and/or ex vivo methodsof immunization of animals (including, e.g., vertebrates and mammals)against at least one virus of the Flaviviridae family, e.g., flavivirus,or variant thereof (including, e.g., a flavivirus that comprises or isrelated to at least one dengue virus of at least one dengue virusserotype or a variant thereof), and/or as a vaccine against at least onevirus of the Flaviviridae family, e.g., flavivirus, or variant thereof,and/or more particularly as a vaccine against a virus of theFlaviviridae family, e.g., flavivirus, that comprises or is related toat least one dengue virus or variant thereof.

[0093] Advantageously, recombinant, synthetic, mutant, and/or isolatedpolypeptides and/or fusion proteins provided by the invention comprisean amino acid sequence that is capable of modulating, inducing,promoting, and/or enhancing a detectable immune response(s), such as theproduction of antibodies that bind to at least one virus of theFlaviviridae family, e.g., flavivirus (e.g., dengue virus), and/or theproduction of at least one type of antigen relating to the Flaviviridaefamily, e.g., flavivirus antigen (including, e.g., at least one type ofdengue virus antigen) in animal cells (typically vertebrate cells, andmore typically and preferably, mammalian cells, such as human andnonhuman primate cells) in vitro in cell culture and/or ex vivo and/orin vivo in a subject or tissue or cells obtained therefrom. Such anamino acid sequence portion of the polypeptide or fusion protein can bereferred to as an “immunogenic amino acid” or “antigenic amino acid” orsimply “the amino acid” or an “immunogen” or “antigen” of the invention.

[0094] A further desirable feature of the recombinant, synthetic,mutant, and/or isolated polypeptides of the invention, and therespective polynucleotides of the invention that encode suchpolypeptides, is the ability to induce, promote, modulate, and/orenhance an immune response(s) to at least one flavivirus, including,e.g., to at least one dengue virus of at least one serotype, andpreferably to at least one dengue virus of each of at least twoserotypes, more preferably to at least one dengue virus of each of atleast three serotypes, and even more preferably to at least one denguevirus of each of at least four known virus dengue virus serotypes (e.g.,DEN-1, DEN-2, DEN-3, and/or DEN-4).

[0095] A further desirable feature of the recombinant synthetic, mutant,and/or isolated polypeptides of the invention, and the polynucleotidesthat encode such polypeptides, is the ability to induce, promote,enhance or modulate a neutralizing antibody response(s) against at leastone flavivirus, preferably against at least one dengue virus of at leastone serotype, more preferably against at least one dengue virus of eachof at least two serotypes, even more preferably against at least onedengue virus of each of at least three serotypes, and most preferablyagainst at least one dengue virus of each of at least the four knownvirus dengue virus serotypes (e.g., DEN-1, DEN-2, DEN-3, and/or DEN-4).

[0096] More particular and desirable characteristics of the recombinant,synthetic, mutant, and/or isolated polypeptides and/or fusion proteinsof the invention, polynucleotides encoding these and other polypeptidesand fusion proteins, and related cells, antibodies, vectors,compositions, diagnostic assays and methods of making and using suchpolypeptides and polynucleotides are described in detail herein.

[0097] The term “PRM15” (or “prM15” and sometimes alternatively referredto as “spM”) when used with reference to an amino acid generally refersto an amino acid sequence typically comprising about 15 amino acidresidues. In some embodiments, the PRM15 amino acid sequence includes,in addition, a methionine (“Met” or “M”) as the first amino acidresidue; in such embodiments, the PRM15 sequence comprises a total ofabout 16 amino acids. Usually, a PRM15 amino acid sequence comprises thelast 15 amino acid residues of the C terminus of a wild-type (WT),recombinant, mutant, or synthetic dengue virus prM protein, or variantthereof; in some such embodiments, the PRM15 amino acid sequence furthercomprises a methionine as the first amino acid residue of the sequenceand thus is about 16 amino acids in length.

[0098] In reference to a nucleotide sequence, “PRM15” generally refersto a nucleotide sequence typically comprising about 15 amino acidresidues. In some embodiments, the PRM15 nucleotide sequence includesthree nucleotides residues that encode a methionine, which threeresidues are positioned as the first three residues of the sequence,followed in sequence order by the codons encoding the remainingapproximately 15 amino acids. Usually, a PRM15 nucleotide sequencecomprises nucleic acid residues that encode approximately the last 15amino acids of the C terminus of a WT, recombinant, mutant, or syntheticdengue virus prM protein or variant thereof. In some such embodiments,the PRM15 nucleotide sequence further includes three residues thatencode a methionine, which are positioned at the beginning of thenucleotide sequence; in such embodiments, the nucleotide sequenceencodes a total of about 16 amino acids.

[0099] The PRM15 amino acid sequence is typically linked to apolypeptide and effectively acts as a signal sequence for transport ofsuch polypeptide in a cell. The PRM15 signal sequence is usually cleavedfrom the polypeptide subsequently during processing. For example, insome embodiments, the PRM15 sequence is linked to a dengue virusenvelope (“E” or “Env”) protein, such as a WT, recombinant, mutant, orsynthetic dengue virus E protein or to an antigenic orimmunogen-encoding fragment (e.g., truncated recombinant, WT, orsynthetic E protein) or variant thereof.

[0100] A variety of PRM15 amino acid signal sequences are describedherein (see, e.g., SEQ ID NOS:52-64). A variety of PRM15 nucleotidesequences are also described herein (see, e.g., SEQ ID NOS:272-284). APRM15 amino acid sequence can be readily determined from a prM proteinsequence by identifying, e.g., approximately the last 15 amino acidresidues of the C terminus of the prM sequence. If desired, a Metresidue can be added at the beginning of the amino acid sequence. APRM15 nucleic acid sequence can be similarly determined by identifyingthe nucleic acid residues that encode approximately the last 15 aminoacid residues of the C terminus of a particular prM sequence; ifdesired, a nucleic acid codon encoding a Met residue can be similarlyadded as the first codon of such nucleic acid sequence. PRM15 amino acidand nucleic acid sequences can be synthesized by using standard proteinand nucleic acid synthesis techniques, respectively, as would be know toone of skill in the art and as described below.

[0101] In one aspect, the invention includes a chimeric polypeptidecomprising a sequence that has at least about 90% amino acid sequenceidentity to a polypeptide sequence comprising from amino acid residue 16to the last amino acid residue of the amino acid sequence of a syntheticor recombinant C15/full prM/full E or PRM15/truncated E polypeptide ofthe invention, and nucleic acid encoding said chimeric polypeptide.

[0102] The term “tE” (or “ETRUNC” or “E-truncated” or the like) whenused with reference to an amino acid sequence refers to a truncated (“t”or “trunc”) envelope (E) protein of a flavivirus. Such flavivirus can bean isolated wild-type, recombinant, mutant, or synthetic flavivirus orvariant thereof. In one aspect, the flavivirus comprises a dengue virus,and the truncated E protein comprises a truncated wild-type dengue virusE protein or a variant thereof, or a truncated recombinant, mutant, orsynthetic dengue virus E protein. Compared to a non-truncated E protein,a truncated E protein lacks one or more amino acid residues of the Cterminus of the non-truncated form of the E protein. When used withreference to a nucleic acid sequence, the term “tE” (or “ETRUNC” or“E-truncated” or the like) refers to a truncated nucleic acid sequencethat corresponds to or encodes a truncated E protein. A nucleic acidsequence that encodes a truncated E protein similarly lacks one or morenucleic acid residues from the C terminus compared to the non-truncatednucleic acid.

[0103] In one embodiment, the truncated dengue envelope (E) protein(DEN-1, DEN-2, DEN-3, or DEN-4) comprises a polypeptide sequencecomprising from about 70% to about 98% (e.g., about 85%, 86%, 87%, 88%,90%, 92%, or 95%) of the contiguous amino acid residues of therespective dengue E protein as measured in sequence order from the Nterminus amino acid residue of the amino acid sequence of the respectivedengue E protein. In other words, the truncated E protein comprises afragment of a dengue E protein sequence; such fragment lacks the aminoacid residues corresponding to from about 2% to about 30% (e.g., about15%, 14%, 13%, 12%, 10%, 8%, or 5%) of the amino acid residues of therespective full-length dengue E protein sequence, as measured from the Cterminus amino acid residue of the full-length dengue E proteinsequence. A nucleic acid sequence encoding such a truncated dengue Eprotein does not include the nucleotide residues encoding from about 2%to about 30% (e.g., about 15%, 14%, 13%, 12%, 10%, 8%, or 5%) of theamino acid residues of the C terminus of the respective full lengthdengue E protein sequence, as measured from the C terminus amino acidresidue of the full length E protein sequence.

[0104] In one particular embodiment, the truncated DEN-1 E proteincomprises a polypeptide sequence comprising from about 87% of thecontiguous amino acid residues of the DEN-1 E protein as measured insequence order from the N terminus amino acid residue, and the nucleicacid sequence encoding such a truncated DEN-1 E protein excludesnucleotide residues encoding about the last 13% of the amino acidresidues of the C terminus of the DEN-1 E protein sequence, as measuredfrom the C terminus amino acid residue.

[0105] In one particular embodiment, the truncated DEN-2 E proteincomprises a polypeptide sequence comprising from about 90% of thecontiguous amino acid residues of the DEN-2 E protein as measured insequence order from the N terminus amino acid residue, and the nucleicacid sequence encoding such a truncated DEN-2 E protein excludesnucleotide residues encoding about the last 10% of the amino acidresidues of the C terminus of the DEN-2 E protein sequence, as measuredfrom the C terminus amino acid residue.

[0106] In one particular embodiment, the truncated DEN-3 E proteincomprises a polypeptide sequence comprising from about 89% of thecontiguous amino acid residues of the DEN-3 E protein as measured insequence order from the N terminus amino acid residue, and the nucleicacid sequence encoding such a truncated DEN-3 E protein excludesnucleotide residues encoding about the last 11% of the amino acidresidues of the C terminus of the DEN-3 E protein sequence, as measuredfrom the C terminus amino acid residue.

[0107] In one embodiment, the truncated DEN-4 E protein comprises apolypeptide sequence comprising from about 90% of the contiguous aminoacid residues of the DEN-4 E protein as measured in sequence order fromthe N terminus amino acid residue, and the nucleic acid sequenceencoding such a truncated DEN-4 E protein excludes nucleotide residuesencoding about the last 10% of the amino acid residues of the C terminusof the DEN-4 E protein sequence, as measured from the C terminus aminoacid residue.

[0108] The term “CO” refers to “codon optimization” or a “codonoptimized” sequence. When used with reference to a nucleic acidsequence, the term refers to a codon optimized nucleic acid sequence.When used with reference to an amino acid sequence, the term refers toan amino acid sequence that corresponds to or is encoded by a codonoptimized nucleic acid sequence.

[0109] The terms “polypeptide,” “protein,” and “peptide,” herein andused throughout synonymously refer to any polymer formed from multipleamino acids associated, at least in part, by covalent bonding (e.g.,“protein” as used herein refers both to linear polymers (chains) ofamino acids associated by peptide bonds as well as proteins exhibitingsecondary, tertiary, or quaternary structure, which can include otherforms of intramolecular and intermolecular association, such as hydrogenand van der Waals bonds, within or between peptide chain(s)), unlessotherwise stated. The term “polypeptide” and “protein” includes fusionproteins, unless otherwise stated.

[0110] The term “recombinant” when used with reference to an amino acid(e.g., peptide, polypeptide, protein, antigen) typically refers to anon-naturally occurring amino acid (i.e., not found in nature) (e.g.,non-naturally occurring peptide, polypeptide, protein, antigen). A“recombinant polypeptide” includes any polypeptide expressed or capableof being expressed from a recombinant nucleic acid (however, therecombinant nucleic acid sequence need not include all of the coding ornucleotide sequence elements necessary for expression), or anypolypeptide comprising an amino acid sequence, wherein each amino acidresidue of the sequence corresponds to or is capable of being encoded bya codon of a nucleic acid sequence, including a recombinant nucleic acidsequence.

[0111] The term “recombinant” when used with reference to a nucleic acid(e.g., a polynucleotide or other nucleotide) typically refers to anon-naturally occurring nucleic acid. When used with reference to anucleic acid, the term “recombinant” may indicate that the nucleic acidhas been modified by the introduction of at least one exogenous (i.e.,foreign, and typically heterologous) nucleotide or the alteration of atleast one native nucleotide component of the nucleic acid. A“recombinant vector” refers to a non-naturally occurring vector,including, e.g., a vector comprising a recombinant nucleic acidsequence.

[0112] In one aspect, a “recombinant polynucleotide” or a “recombinantpolypeptide” is a non-naturally occurring polynucleotide or polypeptide.A recombinant polynucleotide or recombinant polypeptide may includenucleic acids or amino acids, respectively, from more than one sourcenucleic acid or polypeptide, which source nucleic acid or polypeptidecan be a naturally occurring nucleic acid or polypeptide, or can itselfhave been subjected to mutagenesis, alteration, recombination, or othertype of modification. The source polynucleotides or polypeptides fromwhich the different nucleic acid or amino acid sequences are derived aresometimes homologous (i.e., have, or encode a polypeptide that encodes,the same or a similar structure and/or function), and are often fromdifferent isolates, serotypes, strains, species, of organism or fromdifferent disease states, for example.

[0113] The term “recombinant” when used with reference to a cellindicates that the cell comprises a recombinant molecule, such as arecombinant nucleic acid, recombinant polypeptide, or recombinant vector(e.g., non-naturally occurring nucleic acid, polypeptide, or vector). Inone aspect, the term “recombinant” when used with reference to a cellindicates that the cell replicates a heterologous nucleic acid, orexpresses a peptide or protein encoded by a heterologous nucleic acid.Recombinant cells can contain genes that are not found within the native(non-recombinant) form of the cell. Recombinant cells can also containgenes found in the native form of the cell wherein the genes aremodified and re-introduced into the cell by artificial means. The termalso encompasses cells that comprise a nucleic acid endogenous to thecell that has been modified without removing the nucleic acid from thecell; such modifications include, e.g., those obtained by genereplacement, site-specific mutation, and related techniques.

[0114] The term “synthetic” in reference to a molecule or componentmeans an artificial or non-naturally occurring molecule or component,respectively. For example, a synthetic polynucleotide is an artificial,non-naturally occurring polynucleotide. Techniques for syntheticallyproducing a molecule, component, or combination thereof, including,e.g., a synthetic polynucleotide, polypeptide, fusion protein, vector,virus, virus-like particle, cell, composition, and the like, are furtherdescribed herein.

[0115] For ease of readability, recombinant, synthetic, mutant and/orvariant polypeptides, polynucleotides, fusion proteins, vectors, cells,and antibodies of the invention are often referred to simply as“recombinant” polypeptides, polynucleotides, fusion proteins, vectors,cells, and antibodies, respectively.

[0116] The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al. (1991) NucleicAcid Res 19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608;Cassol et al. (1992); Rossolini et al. (1994) Mol Cell Probes 8:91-98).The term nucleic acid is used interchangeably with polynucleotide, and(in appropriate contexts) gene, cDNA, and mRNA encoded by a gene.

[0117] “Nucleic acid derived from a gene” refers to a nucleic acid forwhose synthesis the gene, or a subsequence thereof, has ultimatelyserved as a template. Thus, an mRNA, a cDNA reverse transcribed from anmRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA,an RNA transcribed from the amplified DNA, etc., are all derived fromthe gene and detection of such derived products is indicative of thepresence and/or abundance of the original gene and/or gene transcript ina sample.

[0118] A nucleic acid is “operably linked” with another nucleic acidsequence when it is placed into a functional relationship with anothernucleic acid sequence. For instance, a promoter or enhancer is operablylinked to a coding sequence if it increases the transcription of thecoding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame. However, sinceenhancers generally function when separated from the promoter by severalkilobases and intronic sequences may be of variable lengths, somepolynucleotide elements may be operably linked but not contiguous.

[0119] An “isolated” polypeptide refers to a polypeptide separated fromone or more components and/or the environment with which it is normallyassociated (e.g., other peptides, polypeptides, proteins (includingcomplexes, cellular contaminants, cellular components, etc.), cells,etc.). An “isolated” nucleic acid (or an isolated nucleotide or isolatedpolynucleotide) refers to a nucleic acid (or nucleotide orpolynucleotide) that is isolated from one or more components and/or theenvironment with which it normally associates. Typically, an isolatednucleic acid refers to a nucleic acid that is not immediately contiguouswith one or more nucleic acids with which it is immediately contiguous(i.e., at the 5′ and/or 3′ end) in the sequence from which it isobtained and/or derived.

[0120] Typically, isolation of a component (e.g., polypeptide,polynucleotide, fusion protein, vector, cell) renders it the predominantcomponent present in a composition, mixture, or collection ofcomponents; i.e., on a molar basis it is more abundant than any otherindividual species in the composition. For example, isolation of apolypeptide or polynucleotide renders the polypeptide or polynucleotide,respectively, the predominant molecule or species present in acomposition, mixture, or collection of molecules. Such a “substantiallypure” polypeptide or polynucleotide (or other component), for example,typically forms at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, byweight (typically on a molar basis), of all macromolecular speciespresent in a particular composition. Desirably, the substantially purepolypeptide or polynucleotide exhibits essential homogeneity (i.e.,contaminant species cannot be detected in the composition byconventional detection methods). The term “purified” generally denotesthat a polynucleotide or polypeptide is free or at least substantiallyfree of other components as determined by standard analytical techniques(e.g., forms a band electrophoretic gel, chromatographic eluate, and/ora media subjected to density gradient centrifugation) and/or forms atleast about 80%, at least about 85%, preferably at least about 90%, andmore preferably at least about 95%, of the macromolecular species in aparticular composition.

[0121] The term “subject” as used herein includes, but is not limitedto, an organism; an animal, including a mammal, which includes, e.g., ahuman, non-human primate (e.g., baboon, orangutan, chimpanzee, monkey),mouse, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, horse,monkey, sheep, or other non-human mammal, and a non-mammal, including,e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken orduck) or a fish, and a non-mammalian invertebrate.

[0122] The term “cytokine” includes, for example, interleukins,interferons, chemokines, hematopoietic growth factors, tumor necrosisfactors and transforming growth factors. In general these are smallmolecular weight proteins that regulate maturation, activation,proliferation, and differentiation of cells of the immune system.

[0123] A “variant” of a polypeptide is a polypeptide that differs in oneor more amino acid residues from a parent or reference polypeptide,usually in at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 25, 50, 75, 100 or more amino acid residues.

[0124] A “variant” of a nucleic acid is a nucleic acid that differs inone or more nucleic acid residues from a parent or reference nucleicacid, usually in at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 17, 20, 21, 24, 27, 30, 33, 36, 39, 40, 45, 50, 60, 75, 150,225, 300 or more nucleic acid residues.

[0125] An “antigen” refers to a molecule that is capable of inducing,promoting, enhancing, or modulating an immune response or immunereaction. In some instances, the immune response or immune reaction is ahumoral and/or cellular response. An antigen may induce, promote,enhance or modulate an immune response or immune reaction in cells invitro and/or in vivo in a subject and/or ex vivo in a subject's cells ortissues. Such immune response or reaction may include, but is notlimited to, eliciting the formation of antibodies in a subject, orgenerating a specific population of lymphocytes reactive with theantigen. Antigens are typically macromolecules (e.g., proteins andpolysaccharides) that are foreign to the host.

[0126] A “subsequence” or “fragment” of nucleic acids or amino acidsrefers to a sequence of nucleic acids or amino acids, respectively, thatcomprises any part or segment of a longer sequence of nucleic acids(e.g., polynucleotide) or amino acids (e.g., polypeptide), respectively,up to and including the complete (entire) nucleic acid sequence orcomplete amino acid sequence.

[0127] An “adjuvant” refers to a molecule or substance that augments orenhances an immune response, including, for example, but not limited to,an antigen's immune-stimulating properties or the pharmacologicaleffect(s) of a compound or drug. An adjuvant may non-specificallyenhance an immune response, e.g., the immune response to an antigen.“Freund's Complete Adjuvant,” for example, is an emulsion of oil andwater containing an immunogen, an emulsifying agent and mycobacteria.Another example, “Freund's incomplete adjuvant,” is the same, butwithout mycobacteria. An adjuvant may comprise oils, emulsifiers, killedbacteria, aluminum hydroxide, or calcium phosphate (e.g., in gel form),or combinations thereof. An adjuvant may be administered into a subject(e.g., via injection intramuscularly or subcutaneously) in an amountsufficient to produce antibodies.

[0128] “Naturally occurring” as applied to an object refers to the factthat the object can be found in nature as distinct from beingartificially produced by man. A polypeptide or polynucleotide sequencethat is present in an organism (including viruses, bacteria, protozoa,insects, plants or mammalian tissue) that can be isolated from a sourcein nature and which has not been intentionally modified by man in thelaboratory is naturally occurring. Non-naturally occurring as applied toan object means that the object is not naturally-occurring—i.e., theobject cannot be found in nature as distinct from being artificiallyproduced by man.

[0129] Numbering of a given amino acid polymer or nucleotide polymer“corresponds to numbering” of a selected amino acid polymer or nucleicacid polymer when the position of any given polymer component (e.g.,amino acid residue, nucleotide residue) is designated by reference tothe same or an equivalent residue position in the selected amino acid ornucleotide polymer, rather than by the actual position of the componentin the given polymer. Thus, for example, the numbering of a given aminoacid position in a given polypeptide sequence corresponds to the same orequivalent amino acid position in a selected polypeptide sequence usedas a reference sequence.

[0130] A vector is a component or composition for facilitating celltransduction or transfection by a nucleic acid, or expression of thenucleic acid in the cell. Vectors include, e.g., plasmids, cosmids,viruses, YACs, bacteria, poly-lysine, etc. An “expression vector” or“expression cassette” is a nucleic acid construct or sequence withnucleic acid elements that permit transcription of a nucleic acid in ahost cell and/or that are capable of effecting expression of a nucleicacid in a host compatible with such construct or sequence. An expressionvector or cassette can be generated recombinantly or synthetically bymethods known in the art. The expression vector or cassette can be partof a plasmid, virus, or nucleic acid fragment. The expression vector orexpression cassette typically includes a nucleic acid to be transcribed(e.g., a nucleic acid encoding a desired polypeptide) operably linked toa promoter. The nucleic acid to be transcribed is typically under thedirection or control of the promoter. An expression vector or cassetteoptionally includes transcription termination signal(s). Additionalfactors necessary or helpful in effecting expression may also be used asdescribed herein. For example, an expression vector or cassette can alsoinclude nucleotide sequences that encode a signal sequence that directssecretion of an expressed protein from the host cell. Enhancers andother nucleic acid sequences that influence nucleotide expression orgene expression can also be included.

[0131] “Substantially the entire length of a polynucleotide sequence” or“substantially the entire length of a polypeptide sequence” refers to atleast about 50%, generally at least about 60%, 70%, or 75%, usually atleast about 80% or 85%, or typically at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of a length of apolynucleotide sequence or polypeptide sequence.

[0132] The term “pharmaceutical composition” means a compositionsuitable for pharmaceutical use in a subject, including an animal orhuman. A pharmaceutical composition generally comprises an effectiveamount of an active agent and a carrier, including, e.g., apharmaceutically acceptable carrier.

[0133] The term “effective amount” means a dosage or amount of amolecule or composition sufficient to produce a desired result. Forexample, the desired result may comprise a measurable or testableinduction, promotion, enhancement or modulation of an immune response ina subject to whom a dosage or amount of a particular antigen orimmunogen (or composition thereof) has been administered. In one aspect,the desired result may comprise a measurable or testable induction,promotion, enhancement of an immune response in a subject to whom adosage or amount of a particular viral antigen or immunogen (orcomposition thereof) has been administered sufficient to protect thesubject against challenge by a virus. In another aspect, the desiredresult may comprise an objective or subjective improvement in thesubject receiving a dosage or amount of a particular molecule orcomposition (e.g., the subject to whom the dosage or amount of theparticular molecule or composition is administered).

[0134] A “prophylactic treatment” is a treatment administered to asubject who does not display and/or suffer from signs or symptoms of adisease, pathology, or medical disorder, or displays and/or suffers fromonly early signs or symptoms of a disease, pathology, or disorder, suchthat treatment is administered for the purpose of diminishing,preventing, and/or decreasing the risk of developing the disease,pathology, or medical disorder. A prophylactic treatment functions as apreventative treatment against a disease or disorder. A “prophylacticactivity” is an activity of an agent, such as a nucleic acid, vector,gene, polypeptide, protein, molecule, substance, or composition thereof,that when administered to a subject who does not display and/or sufferfrom signs or symptoms of pathology, disease, or disorder, or whodisplays and/or suffers from only early signs or symptoms of pathology,disease, or disorder, can diminish, prevent, and/or decrease the risk ofthe subject developing such pathology, disease, or disorder. A“prophylactically useful” agent or molecule (e.g., nucleic acid orpolypeptide) refers to an agent or molecule useful in diminishing,preventing, immunizing against, treating, and/or decreasing developmentof a pathology, disease, or disorder.

[0135] A “therapeutic treatment” is a treatment administered to asubject who displays and/or suffers from symptoms or signs of pathology,disease, or disorder, for the purpose of diminishing, treating, and/oreliminating those signs or symptoms of pathology, disease, or disorder.A “therapeutic activity” is an activity of an agent, such as a nucleicacid, vector, gene, polypeptide, protein, molecule, substance, orcomposition thereof, that eliminates, treats, and/or diminishes signs orsymptoms of pathology, disease, or disorder, when administered to asubject displaying and/or suffering from such signs or symptoms. A“therapeutically useful” agent or molecule (e.g., nucleic acid orpolypeptide) is an agent or molecule that is useful in diminishing,treating, and/or eliminating such signs or symptoms of a pathology,disease, or disorder.

[0136] The term “gene” broadly refers to any segment of DNA associatedwith a biological function. Genes include coding sequences and/orregulatory sequences required for their expression. Genes also includenon-expressed DNA nucleic acid segments that, e.g., form recognitionsequences for other proteins (e.g., promoter, enhancer, or otherregulatory regions). Genes can be obtained from a variety of sources,including cloning from a source of interest or synthesizing from knownor predicted sequence information, and may include sequences designed tohave desired parameters.

[0137] Generally, the nomenclature used hereafter and the laboratoryprocedures in cell culture, molecular genetics, molecular biology,nucleic acid chemistry, and protein chemistry described below are thosewell known and commonly employed by those of ordinary skill in the art.Standard techniques, such as described in Sambrook et al., MolecularCloning—A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989 (hereinafter “Sambrook”) andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (1994, supplemented through 1999)(hereinafter “Ausubel”), are used for recombinant nucleic acid methods,nucleic acid synthesis, cell culture methods, and transgeneincorporation, e.g., electroporation, injection, gene gun, impressingonto or through the skin or tissue of a subject, and lipofection.Generally, oligonucleotide synthesis and purification steps areperformed according to specifications. The techniques and procedures aregenerally performed according to conventional methods in the art andvarious general references which are provided throughout this document.The procedures therein are believed to be well known to those ofordinary skill in the art and are provided for the convenience of thereader.

[0138] As used herein, an “antibody” refers to a protein comprising oneor more polypeptides substantially or partially encoded byimmunoglobulin genes or fragments of immunoglobulin genes. The termantibody is used to mean whole antibodies and binding fragments thereof.The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (e.g.,antibody) structural unit comprises a tetramer. Each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 KDa) and one “heavy” chain (about 50-70 KDa). TheN-terminus of each chain defines a variable region of about 100 to 110or more amino acids primarily responsible for antigen recognition. Theterms variable light chain (VL) and variable heavy chain (VH) refer tothese light and heavy chains, respectively.

[0139] Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′2, a dimer of Fab whichitself is a light chain joined to VH-CH1 by a disulfide bond. TheF(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′) 2 dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region. The Fc portion of the antibody molecule correspondslargely to the constant region of the immunoglobulin heavy chain, and isresponsible for the antibody's effector function (see, FundamentalImmunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a moredetailed description of other antibody fragments). While variousantibody fragments are defined in terms of the digestion of an intactantibody, one of skill will appreciate that such Fab′ fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term antibody, as used herein also includesantibody fragments either produced by the modification of wholeantibodies or synthesized de novo using recombinant DNA methodologies.

[0140] Antibodies also include single-armed composite monoclonalantibodies, single chain antibodies, including single chain Fv (sFv)antibodies in which a variable heavy and a variable light chain arejoined together (directly or through a peptide linker) to form acontinuous polypeptide, as well as diabodies, tribodies, and tetrabodies(Pack et al. (1995) J Mol Biol 246:28; Biotechnol 11:1271; andBiochemistry 31:1579). The antibodies are, e.g., polyclonal, monoclonal,chimeric, humanized, single chain, Fab fragments, fragments produced byan Fab expression library, or the like.

[0141] The term “epitope” generally refers to a peptide or polypeptidedeterminant capable of specifically binding to an antibody. Epitopesusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics. Conformational and nonconformational epitopes aredistinguished in that the binding to the former but not the latter islost in the presence of denaturing solvents.

[0142] An “antigen-binding fragment” of an antibody is a peptide orpolypeptide fragment of the antibody that binds or selectively binds anantigen. An antigen-binding site is formed by those amino acids of theantibody that contribute to, are involved in, or affect the binding ofthe antigen. See Scott, T. A. and Mercer, E. I., Concise Encyclopedia:Biochemistry and Molecular Biology (de Gruyter, 3d ed. 1997), andWatson, J. D. et al., Recombinant DNA (2d ed. 1992) [hereinafter“Watson, Recombinant DNA”], each of which is incorporated herein byreference in its entirety for all purposes.

[0143] The term “screening” describes, in general, a process thatidentifies optimal or optimized molecules. Several properties of therespective molecules can be used in selection and screening including,for example, ability to induce a desired immune response in a testsystem. Selection is a form of screening in which identification andphysical separation are achieved simultaneously by expression of aselection marker, which, in some genetic circumstances, allows cellsexpressing the marker to survive while other cells die (or vice versa).Because of limitations in studying primary immune responses in vitro, invivo studies are particularly useful screening methods. In one aspect,screening refers to a process that identifies a polypeptide (or anucleic acid encoding such polypeptide), wherein the polypeptide inducesor is capable of inducing an immune response to at least a portion ofdengue viruses of at least one virus serotype in a subject, or cells ofa subject, that is about equal to or greater than the immune responseinduced or capable of being induced by a reference polypeptide (e.g.,wild-type polypeptide).

[0144] A “specific binding affinity” between two molecules, e.g., aligand and a receptor, means a preferential binding of one molecule foranother in a mixture of molecules. The binding of the molecules istypically considered specific if the binding affinity is about 1×10² M⁻¹to about 1×10¹⁰ M⁻¹ (i.e., about 10⁻²-10⁻¹⁰ M) or greater, includingabout 10⁴ to 10⁶ M⁻¹, about 10⁶ to 10⁷ M⁻¹, or about 10⁸ M⁻¹ to 10⁹ M⁻¹or 10¹⁰ M⁻¹.

[0145] “Avidity” refers to the tendency of an antibody to bind anantigen. The higher the avidity, the greater the affinity of theantibody for the antigen, the greater the binding of the antibody to theantigen, and the greater the stability of the antigen-antibody complexformed by binding of the antibody to the antigen.

[0146] The term “immunoassay” includes an assay that uses an antibody orimmunogen to bind or specifically bind an antigen. The immunoassay maybe characterized by the use of specific binding properties of aparticular antibody to isolate, target, and/or quantify the antigen.

[0147] The invention provides a recombinant, synthetic, mutant, and/orisolated polypeptide comprising an immunogenic amino acid sequence thatis substantially identical (e.g., at least about 75%, 80%, 85%, 86%,87%, 88% or 89%, preferably at least about 90%, 91%, 92%, 93%, or 94%,and more preferably at least about 95% (e.g., about 87-95%), 96% 97%,98%, 99%, 99.5% sequence identity) to an amino acid sequence of at leastone of SEQ ID NOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and 345.In addition, the invention provides a recombinant, synthetic, mutant,and/or isolated polynucleotide that encodes an immunogenic amino acidsequence that is substantially identical to a nucleic acid sequence ofat least one of SEQ ID NOS:156-218, 235, 254-271, 285-330, 342, and 344.As applied to polypeptides, the term “substantial identity” means thattwo or more amino acid sequences, when optimally aligned, such as by GAPor BESTFIT programs using default gap weights, by visual inspection, orany other suitable technique such as the sequence analysis and identityalgorithms further describe herein, share at least about 60%, typicallyat least about 65%, usually at least about 70%, often at least about75%, usually at least about 80%, at least about 85%, about 86%, about87%, about 88%, or about 89%, and preferably at least about 90%, or more(e.g., at least about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, orabout 99.5% or more) amino acid sequence identity. Similarly, as appliedto nucleic acids, the term substantial identity or substantialsimilarity means that the two or more nucleic acid sequences, whenoptimally aligned, such as by the programs BLAST, GAP or BESTFIT usingdefault gap weights (described in detail below along with other suitableprograms and techniques for assessing nucleic acid sequence identitylevels), or by visual inspection, share at least about 60% nucleic acidsequence identity or sequence similarity, at least about 70% or at leastabout 75% sequence identity or sequence similarity, more desirably atleast about 80 or about 85% nucleic acid sequence identity or sequencesimilarity; preferably at least about 90% nucleic acid sequence identityor sequence similarity, and more preferably at least about 95% nucleicacid sequence identity or sequence similarity (including, e.g., about90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, 99, 99.5, or more percentnucleotide sequence identity or sequence similarity).

[0148] “Identity” (sometimes referred to as “overall identity”—incontrast to “local identity,” which is discussed further herein) withrespect to amino acid or nucleotide sequences refers to the percentageof amino acid residues or nucleotide bases, respectively, that areidentical in the two amino acid or nucleotide sequences when two suchamino acid sequences or two such nucleotide sequences are optimallyaligned with one another. If, in the optimal alignment, a position in afirst sequence is occupied by the same amino acid residue or nucleotideresidue as the corresponding position in the second corresponding aminoacid or nucleotide sequence, the sequences exhibit identity with respectto that residue position. The level of identity between two sequences(or “percent sequence identity”) is measured as a ratio of the number ofidentical positions shared by the sequences with respect to the size ofthe sequences analyzed (i.e., percent sequence identity=(number ofidentical positions/total number of positions)×100).

[0149] The “optimal alignment” is the alignment that provides thehighest identity between the aligned sequences. In obtaining the optimalalignment, gaps can be introduced, and some amount of non-identicalsequences and/or ambiguous sequences can be ignored. Preferably, if agap needs to be inserted into a first sequence to achieve the optimalalignment, the percent identity is calculated using only the residuesthat are paired with a corresponding amino acid residue (i.e., thecalculation does not consider residues in the second sequences that arein the “gap” of the first sequence). However, it is often preferablethat the introduction of gaps and/or the ignoring ofnon-homologous/ambiguous sequences are associated with a “gap penalty.”

[0150] While identity between relatively short amino acid or nucleicacid sequences can be easily determined by visual inspection, analysiswith an appropriate algorithm, typically facilitated through computersoftware, commonly is used to determine identity between longersequences. When using a sequence comparison algorithm, test andreference sequences typically are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. A number of mathematical algorithms for rapidly obtainingthe optimal alignment and calculating identity between two or moresequences are known and incorporated into a number of available softwareprograms. Examples of such programs include the MATCH-BOX, MULTAIN, GCG,FASTA, and ROBUST programs for amino acid sequence analysis, and theSIM, GAP, NAP, LAP2, GAP2, and PIPMAKER programs for nucleotidesequences. Preferred software analysis programs for both amino acid andpolynucleotide sequence analysis include the ALIGN, CLUSTALW (e.g.,version 1.6 and later versions thereof), and BLAST programs (e.g., BLAST2.1, BL2SEQ, and later versions thereof). Select examples of which arefurther described in the following paragraphs.

[0151] For amino, acid sequence analysis, a weight matrix, such as theBLOSUM matrixes (e.g., the BLOSUM45, BLOSUM50, BLOSUM62, and BLOSUM80matrixes—as described in, e.g., Henikoff and Henikoff (1992) Proc NatlAcad Sci USA 89:10915-10919), Gonnet matrixes (e.g., the Gonnet40,Gonnet80, Gonnet120, Gonnet160, Gonnet250, and Gonnet350 matrixes), orPAM matrixes (e.g., the PAM30, PAM70, PAM120, PAM160, PAM250, and PAM350matrixes), are used in determining identity. BLOSUM matrixes arepreferred. The BLOSUM50 and BLOSUM62 matrixes are typically mostpreferred. In the absence of availability of such weight matrixes (e.g.,in nucleic acid sequence analysis and with some amino acid analysisprograms), a scoring pattern for residue/nucleotide matches andmismatches can be used (e.g., a +5 for a match and −4 for a mismatchpattern).

[0152] The ALIGN program produces an optimal global (overall) alignmentof the two chosen protein or nucleic acid sequences using a modificationof the dynamic programming algorithm described by Myers and Miller(1988) CABIOS 4:11-17. Preferably, if available, the ALIGN program isused with weighted end-gaps. If gap opening and gap extension penaltiesare available, they are preferably set between about −5 to −15 and 0 to−3, respectively, more preferably about −12 and −0.5 to −2,respectively, for amino acid sequence alignments, and −10 to −20 and −3to −5, respectively, more preferably about −16 and −4, respectively, fornucleic acid sequence alignments. The ALIGN program and principlesunderlying it are further described in, e.g., Pearson et al. (1988) ProcNatl Acad Sci USA 85:2444-48, and Pearson et al. (1990) Methods Enzymol18:63-98.

[0153] Alternatively, and particularly for multiple sequence analysis(i.e., comparison of more than three sequences), the CLUSTALW program(described in, e.g., Thompson, J. D. et al. (1994) Nuc Acids Res22:4673-4680) can be used. In one aspect, Gap open and Gap extensionpenalties are set at 10 and 0.05, respectively. Alternatively oradditionally, the CLUSTALW program is run using “dynamic” (versus“fast”) settings. Preferably, nucleotide sequence analysis with CLUSTALWis performed using the BESTFIT matrix, whereas amino acid sequences areevaluated using a variable set of BLOSUM matrixes depending on the levelof identity between the sequences (e.g., as used by the CLUSTALW version1.6 program available through the San Diego Supercomputer Center(SDSC)). Preferably, the CLUSTALW settings are set to the SDSC CLUSTALWdefault settings (e.g., with respect to special hydrophilic gappenalties in amino acid sequence analysis). The CLUSTALW program andunderlying principles of operation are further described in, e.g.,Higgins et al. CABIOS, (1992) 8(2): 189-91, Thompson et al. (1994)Nucleic Acids Res 22:4673-80, and Jeanmougin et al. (1998) TrendsBiochem Sci 2:403-07.

[0154] Another useful algorithm for determining percent identity is theFASTA algorithm, which is described in Pearson, W.R. & Lipman, D. J.(1988) Proc Natl Acad Sci USA 85:2444. See also, W. R. Pearson (1996)Methods Enzymol 266:227-258. Preferred parameters used in a FASTAalignment of DNA sequences to calculate percent identity are optimized,BL50 Matrix 15: −5, k-tuple=2; joining penalty=40, optimization=28; gappenalty −12, gap length penalty=−2; and width=16.

[0155] Other preferred algorithms include the BLAST and BLAST 2.0algorithms, which facilitate analysis of at least two amino acid ornucleotide sequences, by aligning a selected sequence against multiplesequences in a database (e.g., GenSeq), or, when modified by anadditional algorithm such as BL2SEQ, between two selected sequences.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (NCBI)(http://www.ncbi.nlm.nih.gov/). The BLAST algorithm parameters W, T, andX determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) can be used with a word length (W) of11, an expectation (E) of 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program (e.g., BLASTP2.0.14; June 29, 2000) can be used with a word length of 3 and anexpectation (E) of 10.

[0156] BLAST program analysis also or alternatively is preferablymodified by low complexity filtering programs such as the DUST or SEGprograms, which are preferably integrated into the BLAST programoperations (see, e.g., Wootton et al. (1993) Comput Chem 17:149-63,Altschul et al. (1991) Nat Genet 6:119-29, Hancock et al. (1991) ComputAppl Biosci 10:67-70, and Wootton et al. (1996) Meth Enzymol266:554-71). In such aspects, if a lambda ratio is used, preferredsettings for the ratio are between 0.75 and 0.95, more preferablybetween 0.8 and 0.9. If gap existence costs (or gap scores) are used insuch aspects, the gap existence cost preferably is set between about −5and −15, more preferably about −10, and the per residue gap costpreferably is set between about 0 to −5, more preferably between 0 and−3 (e.g., −0.5). Similar gap parameters can be used with other programsas appropriate. The BLAST programs and principles underlying them arefurther described in, e.g., Altschul et al. (1990) J Mol Biol215:403-10, Karlin and Altschul (1990) Proc Natl Acad Sci USA 87:2264-68(as modified by Karlin and Altschul (1993) Proc Natl Acad Sci USA90:5873-77), and Altschul et al. (1997) Nucl Acids Res 25:3389-3402.

[0157] Another example of a useful algorithm is incorporated in PILEUPsoftware. The PILEUP program creates a multiple sequence alignment froma group of related sequences using progressive, pair-wise alignments toshow relationship and percent sequence identity or percent sequencesimilarity. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle (1987) J Mol Evol 35:351-360, which issimilar to the method described by Higgins & Sharp (1989) CABIOS5:151-153. Preferred parameters for the PILEUP program are: default gapweight (3.00), default gap length weight (0.10), and weighted end gaps.PILEUP is a component of the GCG sequence analysis software package,e.g., version 7.0 (see, e.g., Devereaux et al. (1984) Nuc Acids Res12:387-395).

[0158] Other useful algorithms for performing identity analysis includethe local homology algorithm of Smith and Waterman (1981) Adv Appl Math2:482, the homology alignment algorithm of Needleman and Wunsch (1970) JMol Biol 48:443, and the search for similarity method of Pearson andLipman (1988) Proc Natl Acad Sci USA 85:2444. Computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, and TFASTA) areprovided in the Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, 575 Science Dr., Madison, Wis.

[0159] Several additional commercially available software suitesincorporate the ALIGN, BLAST, and CLUSTALW programs and similarfunctions, and may include significant improvements in settings andanalysis. Examples of such programs include GCG suite of programs andthose available through DNASTAR, Inc. (Madison, Wis.), such asLasergene® and Protean® programs. A preferred alignment method is theJotun Hein method, incorporated within the MegaLine™ DNASTAR package(MegaLine™ Version 4.03) used according to the manufacturer'sinstructions and default values specified in the program.

[0160] Because various algorithms, matrixes, and programs are commonlyused to analyze sequences, amino acid and polynucleotide sequences arepreferably characterized in terms of approximate identities byindicating a range of identity “about” a particular identity (e.g.;+/−10%, more preferably +/−8%, and even more preferably +/−5% of theparticular identity). Nonetheless, an exact identity can be measured byusing only one of the aforementioned programs, such as a BLAST programdescribed herein or the Hein method.

[0161] In one aspect, the invention provides a recombinant, synthetic,and/or isolated polypeptide (which may be simply referred to as thepolypeptide or recombinant polypeptide) which comprises an amino acidsequence that has at least about 90% amino acid sequence identity to anamino acid sequence of at least one of SEQ ID NOS:1-49, 65-116, 139-148,153-155, 236-253, 343, and 345. Desirably, the recombinant polypeptidecomprises a sequence that has at least about 90% amino acid sequenceidentity to at least two sequences selected from the group of SEQ IDNOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and 345. Favorably,the recombinant polypeptide comprises a sequence that has at least about90% amino acid sequence identity to at least five sequences selectedfrom the group of SEQ ID NOS:1-49, 65-116, 139-148, 153-155, 236-253,343, and 345. Advantageously, the recombinant polypeptide comprises asequence that has at least about 90% amino acid sequence identity to atleast about ten, preferably at least about fifteen, and more preferablyat least about twenty sequences selected from the group of SEQ IDNOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and 345. Preferably,the recombinant polypeptide comprises an amino acid sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% identity to at least one,preferably at least five, and more preferably at least about tensequences selected from any of SEQ ID NOS:1-49, 65-116, 139-148,153-155, 236-253, 343, and 345.

[0162] A recombinant polypeptide that comprises an amino acid selectedfrom the group of SEQ ID NOS:1-49, 65-116, 139-148, 153-155, 236-253,343, and 345 is preferred. The polypeptide can comprise any number ofsuitable additional amino acid sequences, such as, e.g., additionalsequences described elsewhere herein (e.g., signal sequence and/orpurification-facilitating epitope tag (e.g., Whitehorn et al.,Biotechnology 13:1215-19 (1995)), or the polypeptide can consistessentially entirely of an amino acid sequence selected from the groupof SEQ ID NOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and 345.

[0163] Alternatively, but more typically additionally, the recombinantpolypeptide can comprise an amino acid sequence that has substantialfunctional homology to the immunogenic amino acid sequence of anypolypeptide of the invention, such as the amino acid sequence of any oneof SEQ ID NOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and 345.“Substantial functional homology” means that the analyzed amino acidsequences share at least about 60%, typically at least about 65%,usually at least about 70%, often at least about 75%, preferably atleast about 80%, more preferably at least about 85%, and even morepreferably at least about 90%, or more (e.g., 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 99.5% or more) functionally homologous residuesin the optimal homology alignment. The “optimal functional homologyalignment” is the alignment that provides the highest level of homologybetween two amino acid sequences, using the principles described abovewith respect to the “optimal alignment.” Conservative amino acid residuesubstitutions involve exchanging a member within one class of amino acidresidues for a residue that belongs to the same class (identical aminoacid residues are considered functionally homologous or conserved incalculating percent functional homology). The classes of amino acids andthe members of those classes are presented in Table 1. TABLE 1 AminoAcid Residue Classes Amino Acid Class Amino Acid Residues AcidicResidues ASP and GLU Basic Residues LYS, ARG, and HIS HydrophilicUncharged Residues SER, THR, ASN, and GLN Aliphatic Uncharged ResiduesGLY, ALA, VAL, LEU, and ILE Non-polar Uncharged Residues CYS, MET, andPRO Aromatic Residues PHE, TYR, and TRP

[0164] An alternative set of conservative amino acid substitutions,delineated by six conservation groups, is provided in Table 2. TABLE 2Alternative Amino Acid Residue Substitution Groups 1 Alanine (A) Serine(S) Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

[0165] More conservative substitutions exist within the above-describedclasses and can be alternatively preferred. An example of conservationgroups for more conservative substitutions include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alamine-valine, and asparagine-glutamine. Additional groups of aminoacids can also be formulated using the principles described in, e.g.,Creighton (1984) Proteins: Structure and Molecular Properties (2d Ed.1993), W. H. Freeman and Company.

[0166] Typically, one or more amino acid residues in the immunogenicamino acid sequence of the polypeptide that are not identical to acorresponding residue in at least one of the immunogenic amino acidsequences disclosed herein, such as, e.g., SEQ ID NOS:1-49 and 153-155,usually differ from the most related immunogenic amino acid sequence(e.g., the most related sequence selected from the group of SEQ IDNOS:1-49 and 153-155) by conservative amino acid substitutions, i.e.,substitutions with one of more of the groups provided in Table 1 orTable 2 above, or, typically, substitutions that are within a singlegroup in each such table). As such, the disclosure of a polypeptide orprotein sequence herein, in conjunction with the above-describedconservation groups, provides an express listing of all conservativelysubstituted polypeptide sequences relating to these sequences.

[0167] Typically, the immunogenic amino acid sequence of the polypeptidealso exhibits substantial weight homology to one of the immunogenicamino acid sequences of the invention, commonly to at least one of thegroup of SEQ ID NOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and345. Desirably, the immunogenic amino acid sequence has substantiallyhigh weight homology with at least 5, preferably at least about 10, ormore, of the disclosed immunogenic amino acid sequences of the invention(e.g., about 5, 10 or more sequences selected from any of theabove-referenced group of sequences). “Substantial weight homology”means that at least about 60%, preferably at least about 70%, and morepreferably at least about 80% (e.g., about 65-85%), or more (e.g., about87%, 90%, 92%, 95%, or 99%) of the non-identical amino acid residues ata position in the polypeptide are members of the same weight-based “weakconservation group” or “strong conservation group” as the correspondingamino acid in the most identical or functionally homologous sequenceamong the disclosed immunogenic amino acid sequences of the invention,such as an amino acid sequence selected from the group of and SEQ IDNOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and 345. Strong groupconservation is preferred. Weight-based conservation is determined onthe basis of whether the non-identical corresponding amino acid isassociated with a positive score on one of the weight-based matricesdescribed herein (e.g., the BLOSUM50 matrix and preferably the PAM250matrix). Weight-based strong conservation groups include Ser Thr Ala,Asn Glu Gin Lys, Asn His Gin Lys, Asn Asp Glu Gin, Gin His Arg Lys, MetIle Leu Val, Met Ile Leu Phe, His Tyr, and Phe Tyr Trp. Weight-basedweak conservation groups include Cys Ser Ala, Ala Thr Val, Ser Ala Gly,Ser Thr Asn Lys, Ser Thr Pro Ala, Ser Gly Asn Asp, Ser Asn Asp Glu GinLys, Asn Asp Glu Gin His Lys, Asn Glu Gin His Arg Lys, Phe Val Leu IleMet, and His Phe Tyr. The CLUSTALW sequence analysis program providesanalysis of weight-based strong conservation and weak conservationgroups in its output, and offers the preferred technique for determiningweight-based conservation, preferably using the CLUSTALW defaultsettings used by the SDSC.

[0168] Alternatively, but typically in addition to either substantialidentity or substantial functional homology, the polypeptide comprisesan immunogenic amino acid sequence that shares a similar hydropathyprofile (or exhibits similar hydrophilicity) to at least one (preferablyat least 5, and more preferably at least about 10) of the immunogenicamino acid sequences disclosed herein, such as amino acid sequencesselected from the group of SEQ ID NOS:1-49. A hydropathy profile can bedetermined using the Kyte & Doolittle index, the scores for eachnaturally occurring amino acid in the index being as follows: I (+4.5),V (+4.2), L (+3.8), F (+2.8), C (+2.5), M (+1.9); A (+1.8), G (−0.4), T(−0.7), S (−0.8), W (−0.9), Y (−1.3), P (−1.6), H (−3.2); E (−3.5), Q(−3.5), D (−3.5), N (−3.5), K (−3.9), and R (−4.5) (see, e.g., U.S. Pat.No. 4,554,101 and Kyte & Doolittle, (1982) J Molec Biol 157:105-32 forfurther discussion). Preferably, at least about 45%, preferably at leastabout 60%, and more preferably at least about 75% (e.g., at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97% at least about 98%, or at least about 99%) ofthe amino acid residues in the immunogenic amino acid sequence that arenot identical to the corresponding residues in the most identical orfunctionally homologous immunogenic amino acid sequence disclosed herein(“most related homolog”), which homolog is preferably selected from anyof SEQ ID NOS:1-49, exhibit less than a +/−2 change in hydrophilicity,more preferably less than a +/−1 change in hydrophilicity, and even morepreferably less than a +/−0.5 change in hydrophilicity with respect tothe non-identical amino acid residue at the corresponding position inthe most related homolog. Overall, the polypeptide desirably exhibits atotal change in hydrophilicity, with respect to its most related homologselected from the group of SEQ ID NOS:1-49, 65-116, 139-148, 153-155,and 236-253, of less than about 150, more preferably less than about100, and even more preferably less than about 50 (e.g., less than about30, less than about 20, or less than about 10). Examples of typicalamino acid substitutions that retain similar or identical hydrophilicityinclude arginine-lysine substitutions, glutamate-aspartatesubstitutions, serine-threonine substitutions, glutamine-asparaginesubstitutions, and valine-leucine-isoleucine substitutions. Algorithmsand software, such as the GREASE program available through the SDSC,provide a convenient way for quickly assessing the hydropathy profile ofa peptide fragment or peptide portion.

[0169] The polypeptide desirably comprises a substantially identical(e.g., having at least about 75%, 80%, 85%, 86%, 87%, 88% or 89%,preferably at least about 90%, 91%, 92%, 93%, or 94%, and morepreferably at least about 95% (e.g., about 87-95%), 96% 97%, 98%, 99%,99.5% sequence identity), or at least substantially functionallyhomologous, immunogenic amino acid sequence to at least one sequence(preferably at least 5 sequences, and more preferably at least about 10sequences) selected from the group of SEQ ID NOS:1-49, 65-116, 139-148,153-155, and 236-253, wherein at least about 90%, preferably at leastabout 95%, and more preferably 100% of the amino acid residues in thecomposition have a Kyte & Doolittle hydropathy score of above 0, andmore preferably of at least about 1.

[0170] Recombinant Truncated E Protein and Full Length E ProteinPolypeptides

[0171] The invention also provides a recombinant polypeptide comprisingan amino acid sequence that has that has at least about 75%, 80%, 85%,86%, 87%, 88% or 89%, preferably at least about 90%, 91%, 92%, 93%, or94%, and more preferably at least about 95% (e.g., about 87-95%), 96%97%, 98%, 99%, 99.5%, or more amino acid sequence identity to an aminoacid sequence of at least one of SEQ ID NOS:1-49 and 153-155. Such apolypeptide is usually referred to as a recombinant truncated envelope(E) protein polypeptide (or “truncated E” or “tE” polypeptide) asdescribed above. The invention also provides recombinant E polypeptidesof the invention that have a sequence length equivalent to orsubstantially equivalent to (e.g., within about 85%, 87%, 88%, 90%, 92%or more of) the length of the amino acid sequence of an envelope proteinof a wild-type flavivirus, e.g., preferably a dengue virus envelopeprotein. Such a polypeptide is usually referred to as a recombinant fulllength E protein polypeptide (“full length E,” “fill E” or “E”polypeptide).

[0172] In another aspect, the invention provides a polypeptide thatcomprises an amino acid sequence that is substantially identical to atleast one of SEQ ID NOS:2, 3, 5, 25, 29, and 44-46. Such a polypeptidecomprises an amino acid sequence that has at least about 75%, 80%, 85%,86%, 87%, 88% or 89%, preferably at least about 90%, 91%, 92%, 93%, or94%, and more preferably at least about 95% (e.g., about 87-95%), 96%97%, 98%, 99%, 99.5%, or more amino acid sequence identity with at leastone of SEQ ID NOS:2, 3, 5, 25, 29, and 44-46. Desirably, the polypeptidecomprises an amino acid sequence that has at least about 85%, at leastabout 90%, at least about 95%, or more amino acid sequence identity with3, 5, or more sequences selected from any of SEQ ID NOS:2, 3, 5, 25, 29,and 44-46. In one preferred aspect, the polypeptide can comprise,consist essentially of, or consist entirely of an amino acid sequenceaccording of any one of SEQ ID NOS:2, 3, 5, 25, 29, and 44-46.

[0173] In a particular aspect, the invention provides a polypeptide thatcomprises an immunogenic amino acid sequence that is substantiallyidentical (e.g., having at least about 75%, 80%, 85%, 86%, 87%, 88% or89%, preferably at least about 90%, 91%, 92%, 93%, or 94%, and morepreferably at least about 95% (e.g., about 87-95%), 96% 97%, 98%, 99%,99.5% sequence identity) to SEQ ID NOS:2, 5 or 25. Desirably, the aminoacid sequence has at least about 85%, more preferably at least about90%, and even more preferably at least about 95%, or more (e.g., 97%,98%, or 99.5%) sequence identity with SEQ ID NO:5. The inventionincludes a polypeptides comprising SEQ ID NO:2, 5, or 25.

[0174] Some such recombinant truncated E polypeptides or recombinantfull length E polypeptides of the invention polypeptide induce, promote,or enhance an immune response in a subject (e.g., mammal), or populationof cells of a subject, against at least one dengue virus of at least oneserotype selected from the group of dengue-1, dengue-2, dengue-3, anddengue-4. Some such polypeptides induce an immune response in a subjectagainst at least one dengue virus of each of at least two, three, orfour serotypes selected from the group of dengue-1, dengue-2, dengue-3,and dengue-4.

[0175] Some such recombinant truncated E or full length E polypeptidesinduce an immune response in a subject at least one dengue virus of atleast one serotype selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4 that is about equal to or greater than an immuneresponse induced in the subject against the at least one dengue virus ofthe at least one serotype by a WT truncated E protein of each said atleast one dengue virus, wherein said WT truncated E protein has an aminoacid sequence length substantially equivalent to that of the recombinantor synthetic polypeptide. The WT truncated E proteins of dengue-1,dengue-2, dengue-3, and dengue-4 can comprise the amino acid sequencesconsisting essentially of SEQ ID NO:338, 339, 340, and 341,respectively.

[0176] Some such polypeptides induce an immune response in a subject, orpopulation of cells of the subject, against at least one dengue virus ofeach of at least two or three serotypes that is about equal to orgreater than an immune response induced in the subject or cells by a WTtruncated E protein of at least one dengue virus of each of the at leasttwo or three serotypes, respectively, against at least one dengue virusof each of said at least three serotypes.

[0177] Preferably, a recombinant truncated E polypeptide induces animmune response in the subject or cells thereof against at least onedengue virus of each of the four serotypes that is about equal to orgreater than an immune response induced in the subjects or its cells byany of SEQ ID NOS:338-341 against at least one dengue virus of each ofthe four serotypes.

[0178] Some such recombinant truncated E and full length E polypeptidesinduce production of one or more antibodies that bind to at least onedengue virus of at least one dengue virus serotype. Preferably, such apolypeptide induce production of one or more antibodies that bind to atleast one dengue virus of each of at least two, more preferably three,or even more preferably four serotypes. In one particular aspect, arecombinant truncated E or full length E polypeptide induces productionof a number of antibodies that bind to at least one dengue virus of atleast one, two, three, or serotypes that is about equal to or greaterthan the number of antibodies induced by a wild-type truncated E proteinor full length E protein of the at least one dengue virus of the atleast one serotype, respectively. In a preferred aspect, a recombinanttruncated E or full length E polypeptide induces production of a numberof antibodies that bind to at least one dengue virus of each of at leastone, preferably at least two, more preferably at least three, and evenmore preferably at least four serotypes that is about equal to orgreater than the number of antibodies induced by a wild-type truncated Eprotein or full length E protein of the at least one dengue virus ofeach of the at least one, two, three or four serotypes, respectively.

[0179] Some such recombinant truncated E and full length E polypeptidesinduce the production of one or more antibodies that bind morespecifically to at least one dengue virus of the at least one serotypethan is induced by a wild-type truncated E or full length E protein,respectively, of the at least one dengue virus of the at least oneserotype.

[0180] In another aspect, some such recombinant truncated E or fulllength E polypeptide of the invention induce or produce a titer ofneutralizing antibodies against at least one dengue virus of each of atleast one, preferably at least two, more preferably at least three, andeven more preferably at least four dengue virus serotypes. Some suchpolypeptides induce or produce a titer of neutralizing antibodiesagainst at least one dengue virus of at least one serotype that is aboutequal to or greater than a titer of neutralizing antibodies producedagainst the at least one dengue virus of the at least one serotype by awild-type truncated E protein of the at least one dengue virus of the atleast one serotype, wherein each said wild-type truncated E protein isselected from the group of SEQ ID NOS:338-341. Some such polypeptidesinduce or produce a titer of neutralizing antibodies against at leastone dengue virus of each of at least two, at least three, or at leastfour serotypes that is about equal to or greater than a titer ofneutralizing antibodies produced against the at least one dengue virusof each of the at least two, at least three, or at least four serotypesby a wild-type truncated E protein of the at least one dengue virus ofeach of the at least two, at least three, or at least four serotypes,respectively, wherein each said wild-type truncated E protein isselected from the group of SEQ ID NOS:338-341.

[0181] The invention provides recombinant truncated E polypeptides andrecombinant full length E proteins having any of the aforementionedcharacteristics and characteristics described herein (or any combinationof such characteristics), in addition to polypeptides having theadditional or alternative characteristics attendant polypeptides of theinvention as described further herein.

[0182] A polypeptide of the invention can comprise an immunogenic aminoacid sequence of any length (e.g., about 10-1500 amino acids, moretypically about 50-1000 amino acids, and even more frequently about100-800 amino acids). Typically, the immunogenic amino acid sequence ofthe invention is at least about 100, more typically at least about 150,frequently about 200, more frequently about 250, usually at least about300, more usually at least about 350, and even more usually (andtypically preferably) at least about 400 amino acids in length (e.g.,about 400-750 amino acids in length, more typically about 425-685 aminoacids in length for recombinant truncated E protein polypeptides, andcommonly about 425 to about 450 amino acids in length for recombinanttruncated E protein polypeptides of the invention, about 435 to about460 or about 465 for recombinant PRM15/truncated E polypeptides of theinvention, and about 650 to about 680 or about 700 amino acids in lengthfor C15/full prM/full E polypeptides of the invention, as are furtherdiscussed above and below).

[0183] Signal Peptide Sequences

[0184] Recombinant truncated E polypeptides and recombinant full lengthE polypeptides of the invention can also or alternatively comprise anysuitable number and type of additional amino acid sequences, such as oneor more peptide fragments. In one embodiment, for example, suchtruncated E or full length E polypeptide further comprises a signalpeptide. Generally, the signal peptide directs the recombinant orsynthetic polypeptide to the endoplasmic reticulum when the recombinantor synthetic polypeptide is expressed in an animal cell. The inclusionof a signal sequence, which typically directs organelle traffickingand/or secretion of at least a portion of the polypeptide uponexpression in a cell is particularly preferred. Such sequences aretypically present in the immature (i.e., not fully processed) form ofthe polypeptide, and are subsequently removed/degraded by cellularproteases to arrive at the mature form of the protein. For example, thetruncated E or full length E polypeptide can include any suitable signalsequence or combinations of signal sequences that direct the polypeptideto intracellular compartments, such as a sequence that directs thepolypeptide to be transported (e.g., translocated) into (preferably suchthat the protein is processed by and released from) the endoplasmicreticulum or secretory pathway (e.g., the ER, golgi, and other secretoryrelated organelles and cellular compartments), the nucleus, and/or whichdirects the polypeptide to be secreted from the cell, translocated in acellular membrane, or target a second cell apart from the cell theprotein is secreted from. In this respect, the polypeptide can includean intracellular targeting sequence (or “sorting signal”) that directsthe polypeptide to an endosomal and/or lysosomal compartment(s) or othercompartment rich in MHC II to promote CD4+ and/or CD8+ T cellpresentation and response, such as a lysosomal/endosomal-targetingsorting signal derived from lysosomal associated membrane protein 1(e.g., LAMP-1—see, e.g., Wu et al. Proc. Natl. Acad. Sci. USA 92:1161-75(1995) and Ravipraskash et al., Virology 290:74-82 (2001)), a portion orhomolog thereof (see, e.g., U.S. Pat. No. 5,633,234), or other suitablelysosomal, endosomal, and/or ER targeting sequence (see, e.g., U.S. Pat.No. 6,248,565). In some aspects, it may desirable for the intracellulartargeting sequence to be located near or adjacent to a proven/identifiedanti-dengue virus T-cell epitope sequence(s) within the polypeptide,which can be identified by techniques known in the art and describedherein, thereby increasing the likelihood of T cell presentation ofpolypeptide fragments that comprise such epitope(s). Preferably, suchpolypeptides are expressed from recombinant, synthetic, mutant and/orisolated DNA or RNA delivered to a host cell by one or more of thenucleotide or viral nucleotide transfer vectors, including, e.g., one ormore of the gene transfer vectors, described further herein.

[0185] Preferably, the polypeptide comprises a signal sequence thatdirects the polypeptide to the endoplasmic reticulum (ER) (e.g.,facilitates ER translocation of the polypeptide) when the polypeptide isexpressed in a mammalian cell. The polypeptide can comprise any suitableER-targeting sequence. Many ER-targeting sequences are known in the art.Examples of such signal sequences are described in U.S. Pat. No.5,846,540. Commonly employed ER/secretion signal sequences include theSTII or Ipp signal sequences of E. coli, yeast alpha factor signalsequence, and mammalian viral signal sequences such as herpes virus gDsignal sequence. Further examples of signal sequences are described in,e.g., U.S. Pat. Nos. 4,690,898, 5,284,768, 5,580,758, 5,652,139, and5,932,445. Suitable signal sequences can be identified using skill knownin the art. For example, the SignalP program (described in, e.g.,Nielsen et al. (1997) Protein Engineering 10:1-6), which is publiclyavailable through the Center for Biological Sequence Analysis athttp://www.cbs.dtu.dk/services/SignalP, or similar sequence analysissoftware capable of identifying signal-sequence-like domains can beused. Related techniques for identifying suitable signal peptides areprovided in Nielsen et al., Protein Eng. 10(1): 1-6 (1997). Sequencescan be manually analyzed for features commonly associated with signalsequences, as described in, e.g., European Patent Application 0 621 337,Zheng and Nicchitta (1999) J Biol Chem 274(51): 36623-30, and Ng et al.(1996) J Cell Biol 134(2):269-78.

[0186] Recombinant truncated polypeptides having the above-describedcharacteristics typically comprise an immunogenic amino acid sequencethat is shorter in amino acid length than the dengue virus envelopeprotein; that is, the immunogenic amino acid sequence comprises one ormore residues less than the total number of residues of a dengue virusenvelope protein. Particularly, the immunogenic amino acid of suchproteins is typically about 65-95%, and more typically about 80-90%, ofthe size of a dengue virus envelope protein (determined by number ofresidues in the respective proteins), preferably in combination with anER-targeting signal sequence that usually has a size equal to about5-20% of a dengue virus prM sequence (useful fragments of such aminoacid sequences also provided by the invention are discussed furtherherein). Such truncated E polypeptides often lack at least a portion ofthe dengue virus E protein C-terminal transmembrane sequence or afunctional and/or structural homolog thereof. Proteins having suchcharacteristics can exhibit different secretion qualities than a proteinthat comprise such a sequence.

[0187] The invention also provides recombinant polypeptides comprisingan immunogenic amino acid sequence that is equivalent to or similar inlength to a complete or full length flavivirus envelope protein,preferably a dengue virus envelope protein. Such amino acid sequencescan be referred to as “partial full length” and “full length” Epolypeptide sequences, respectively, in contrast to the above-described“truncated” E sequences that comprise an immunogenic amino acid sequencethat is equivalent to or similar in length to a truncated envelopeprotein of a flavivirus, such as a dengue virus. In addition, asdescribed below, the invention also provides polypeptides comprisingsuch a full length immunogenic sequence that further includes a signalpeptide sequence.

[0188] In one embodiment, a recombinant truncated E polypeptide or fulllength E polypeptide of the invention further comprises a signal peptidesequence, wherein the signal peptide sequence comprises, or consistsessentially of, an amino acid sequence of at least about 10 (e.g., about8-20) amino acid residues in length that has at least about 50%(preferably at least about 60%, 65%, 70%, 80%, 85%, 90%, 95%, 98%, ormore) and even more preferably at least about 85-95% amino acid sequenceidentity to the C-terminal 5-20% of a flavivirus prM protein sequence.For example, in one aspect, such signal peptide comprises the last 15amino acid residues of the C terminus of the flavivirus prM proteinsequence (e.g., dengue prM protein sequence). Any suitable flavivirusC-terminal prM sequence can be used as the basis for a signal peptide.The flaviviruses are discussed in, e.g., FIELDS VIROLOGY, supra,VIROLOGY, B. N. Fields et al., eds., Raven Press, Ltd., New York (3rded., 1996 and 4th ed., 2001) and the ENCYCLOPEDIA OF VIROLOGY, R. G.Webster et al., eds., Academic Press (2nd ed., 1999). Several flaviviralprM sequences also are known (see, e.g., Despres et al. (1990) Virus Res16(1):59-75, Venugopal et al. (1995) Vaccine August; 13(11):1000-5,International Patent Application WO 01/39802, and GenBank Accession Nos.AAK97602, AAD28623, GNWVTB, BAA23792, BAA23784, BAA08221, BAA08220 andAAF34187). The C-terminal portions of these and other flavivirus prMsignal sequences or substantially identical homologs (e.g., having atleast about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity)thereof can be generated by standard DNA synthesis techniques (homologscan be generated through directed mutagenesis, recursive sequencerecombination, rational sequence design, or any other suitabletechnique, examples of which are discussed further herein) and fused tosequences encoding the amino acid sequence (e.g., a sequence encodingthe C-terminal-most 10-20 amino acids of a yellow fever virus, orhomolog thereof, can be fused to a sequence encoding any one of SEQ IDNOS:1-49 and 153-155). Introduction of a start codon to the 5′ end ofsuch a prM sequence typically adds an N-terminal methionine to the aminoacid sequence when expressed in a mammalian cell (other modificationsmay occur in bacterial and/or other eukaryotic cells, such asintroduction of an formyl-methionine residue at a start codon). Theinventors contemplate the production and use of such N-terminalmethionine sequences in most aspects where the polypeptide comprises orconsists essentially of the amino acid sequence or at least theN-terminus thereof. Standard nucleic acid synthesis techniques are knownin the art (see, e.g., Beaucage and Caruthers, Tetrahedron Let22:1859-1869 (1981), Mathers et al., EMBO J. 3:801-805 (1984), Saiki etal., Science 239:487-491 (1988) U.S. Pat. No. 4,683,202, and otherreferences cited herein).

[0189] In one aspect, the recombinant truncated E polypeptide or fulllength E polypeptide further comprises a signal peptide, which signalpeptide comprises a signal sequence of about 5-25 amino acids (e.g.,about 15 amino acids, typically about 10-20 amino acids) that has atleast about 50% or at least about 60%, preferably at least about 70%,80%, or 85% (e.g., at least about 65 to about 95%), and more preferablyat least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ormore amino acid sequence identity to an amino acid sequence comprisingthe C-terminal most 15 amino acids of the prM protein selected from thegroup of DEN-1 prM, DEN-2 prM, DEN-3 prM, and DEN-4 prM or to an aminoacid sequence comprising a methionine residue following by theC-terminal most 15 amino acids of the prM protein selected from thegroup of DEN-1 prM, DEN-2 prM, DEN-3 prM, and DEN-4 prM (SEQ IDNOS:52-55, respectively). Such amino acid sequences that comprise amethionine residue followed by 15 amino acid residues are typicallytermed a “PRM15” sequence. In another aspect, the signal peptidecomprises a signal sequence that comprises an amino acid sequence ofsimilar size that exhibits at least about 55% amino acid sequenceidentity (preferably at least about 65%, more preferably at least about75%, and even more preferably at least about 85%, at least about 90%, atleast about 95% (e.g., about 80-99%) to at least one of SEQ IDNOS:52-64. In one aspect, the signal peptide comprises a signal sequenceselected from any of SEQ ID NOS:52-64. The positioning of the signalsequence within the recombinant truncated E polypeptide or full length Epolypeptide depends on the type of signal sequence used. Thefunctionality of signal sequences is often position dependent, withrespect to the remainder of the protein. Preferably, the signal peptidesequence is positioned N-terminal to the immunogenic E polypeptidesequence, particularly where the signal sequence is a flaviviral prMsequence or homolog thereof. The signal peptide sequence can beincorporated in any suitable portion of the polypeptide that allows thesignal sequence to carry out its desired targeting function. Typicallyand preferably, the signal peptide sequence is positioned near to (e.g.,within about 20 amino acids or less) the recombinant immunogenic aminoacid sequence (e.g., the truncated E polypeptide or full length Epolypeptide), or, more desirably, is directly fused to the N-terminus ofthe immunogenic amino acid sequence. In some instances, otherheterologous domains or linkers can be positioned between the signalsequence and the immunogenic E polypeptide. Inclusion of such elementsis further discussed elsewhere herein.

[0190] PRM15/Truncated E Polypeptides and PRM15/Full Length EPolypeptides

[0191] Recombinant E truncated polypeptides comprising a dengue virusprM signal peptide sequence or homolog thereof (e.g., signal peptidesequence of any of SEQ ID NOS:52-64) are typically designated signalpeptide/truncated E polypeptides, or signal peptide/full length Epolypeptides. In a particular format, where the signal peptidescomprises the 15 C-terminal amino acid residues of a flavivirus (e.g.,dengue virus) prM protein, a recombinant polypeptide comprising suchsignal peptide and either a recombinant truncated E polypeptide or fulllength E polypeptide of the invention is termed, respectively, aPRM15/truncated E protein polypeptide (also termed “PRM15/truncated E”polypeptide or “PRM15/tE” polypeptide) or a PRM15/full length E proteinpolypeptide (also termed “PRM15/fill length E” polypeptide or“PRM15/full E” polypeptide). The PRM15 sequence is typically fused tothe first amino acid of the truncated E or full length E polypeptide. Inaddition, PRM15/tE and PRM15/full length E polypeptide sequencestypically include a methionine residue as the first amino acid of thesequence.

[0192] Polypeptides of the invention comprising a recombinant truncatedE polypeptide or full length E polypeptide of the invention and a signalsequence desirably comprise an amino acid sequence that has at leastabout 65%, at least about 70%, preferably at least about 80%, preferablyat least about 85%, 87%, or 89%, more preferably at least about 90%,91%, 92%, 93%, or 94%, and even more preferably at least about 95% aminoacid sequence identity to at least one of SEQ ID NOS:65-116 (suchidentity taking into account both the prM fragment or portion, such as,e.g., PRM15 fragment, and the remaining, immunogenic amino acid sequenceof the truncated E polypeptide). In one embodiment, such a polypeptidecomprises an amino acid that has at least about 75% (e.g., about 80% to100%), desirably at least about 85%, favorably at least about 90%, andmore desirably at least about 95% amino acid sequence identity with atleast one of SEQ ID NOS:66, 67, 69, 89, 93, and 108-110. Preferably, thepolypeptide comprises an amino acid sequence selected from the group ofSEQ ID NOS:65-116 or an amino acid sequence that is at least about 95%,98%, 99%, or more identical with of SEQ ID NOS:65-116. Even morepreferably, the polypeptide comprises an amino acid sequence selectedfrom (or having at least about 98%, about 99%, or more identity with)SEQ ID NOS:66, 67, 69, 89, 93, and 108-110.

[0193] In another aspect, the invention provides a recombinant orsynthetic polypeptide comprising an amino acid sequence that has atleast about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5%, or more amino acidsequence identity to the amino acid sequence of at least one of SEQ IDNOS:65-116, wherein recombinant or synthetic polypeptide induces animmune response in a subject against at least one dengue virus of atleast one serotype selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4.

[0194] Some such PRM15/truncated E or PRM15/full length E polypeptidesinduce an immune response in a subject, or population of cells of thesubject, against at least one dengue virus of at least one serotypeselected from the group of dengue-1, dengue-2, dengue-3, and dengue-4that is about equal to or greater than an immune response inducedagainst the at least one dengue virus of the at least one serotype by awild-type PRM15/truncated E or wild-type PRM15/full E polypeptide,respectively, of the at least one serotype. In one aspect, eachwild-type PRM15/truncated E protein polypeptide is selected from SEQ IDNOS:149-152. Some such PRM15/truncated E or PRM15/full E polypeptidesinduce an immune response in a subject or its cells against at least onedengue virus of each of at least two, at least three, or preferably atleast four serotypes that is about equal to or greater than an immuneresponse induced in the subjects or cells thereof against the at leastone dengue virus of each of the at least two, at least three, or atleast four serotypes, respectively, by a wild-type PRM15/truncated Epolypeptide or wild-type PRM15/full E polypeptide of each of the atleast two, at least three, or at least four serotypes, respectively.Some such PRM15/tE polypeptides comprise an amino acid sequence that hasat least about 90% amino acid sequence identity to the sequence of atleast one of SEQ ID NOS:66, 67, 69, 89, 93, and 108-110. In oneparticular aspect, the polypeptide comprises an amino acid sequenceselected from the group of SEQ ID NOS:66, 67, 69, 89, 93, and 108-110.

[0195] Some such PRM15/tE and full E polypeptides induces production ofone or more antibodies that bind to at least one dengue virus of atleast 1, at least 2, preferably at least 3, and more preferably at least4 dengue virus serotypes (e.g., dengue-1, dengue-2, dengue-3, and/ordengue-4). In one aspect, the number of antibodies produced by aPRM15/truncated E polypeptide that bind to at least one dengue virus ofat least one serotype equal to or greater than the number of antibodiesinduced by a WT PRM15/truncated E protein (also referred to as“PRM15/truncated E polypeptide”) of the at least one serotype, whereineach WT PRM15/truncated E protein polypeptide is selected from SEQ IDNOS:149-152.

[0196] Some such PRM15/tE polypeptides, which typically comprise anamino acid sequence that has at least about 90% sequence identity to thesequence of at least one of SEQ ID NOS:65-116, induce production of anumber of antibodies that bind to at least one dengue virus of each ofthe at least two or three serotypes that is about equal to or greaterthan is induced by a wild-type PRM15/truncated E fusion protein of eachof the at least two or three serotypes, wherein each wild-typePRM15/truncated E fusion protein of the selected serotype is selectedfrom SEQ ID NOS:149-152. For some such polypeptides, the induced numberof antibodies that bind to at least one dengue virus of each of the atleast four serotypes is about equal to or greater than is induced by anyof SEQ ID NOS:149-152.

[0197] Furthermore, some such PRM15/tE polypeptide induces production ofone or more antibodies that bind more specifically to at least onedengue virus of at least one serotype than is induced by a wild-typePRM15/truncated E polypeptide of the at least one dengue virus of the atleast one serotype, wherein the particular wild-type PRM15/truncated Efusion protein is selected from SEQ ID NOS:149-152.

[0198] In another aspect, recombinant PRM15/tE polypeptides andPRM15/full length E polypeptides of invention induce or produce a titerof neutralizing antibodies against at least one dengue virus of at leastone dengue virus serotype, preferably at least one dengue virus of eachof at least two serotypes, more preferably at least one dengue virus ofeach of at least three serotypes, and even more preferably at least onedengue virus of each of at least four serotypes selected from dengue-1,dengue-2, dengue-3, and dengue-4.

[0199] In one aspect of the invention, the titer of neutralizingantibodies that is about equal to or greater than a titer ofneutralizing antibodies produced by the PRM15/tE or PRM15/tE polypeptideagainst at least one dengue virus of at least one serotype at least isequal or greater than that produced by a wild-type PRM15/truncated Epolypeptide or wild-type PRM15/full E polypeptide of the at least onedengue virus of the at least one serotype.

[0200] In one particular aspect, for some such PRM15/tE polypeptides,the titer of antibodies induced against at least one dengue virus ofeach of at least two, three, or four dengue virus serotypes is equal toor greater than against at least one dengue virus of at least oneserotype at least is equal or greater than that induced by a wild-typePRM15/truncated E polypeptide of the same dengue virus of each of the atleast two, three, or four serotypes, wherein the particular wild-typePRM15/truncated E polypeptide of the same serotype (for comparison)fusion protein is selected from SEQ ID NOS:149-152.

[0201] Some such PRM15/tE polypeptides induce at least one neutralizingantibody response in a mammal to or against at least one dengue virus ofeach of at least two, three or four serotypes without an occurrence ofantibody-dependent enhancement (ADE) upon contact of the mammal with theat least dengue virus of each of the at least two, three, or fourserotypes, respectively.

[0202] In one aspect of the invention, a recombinant polypeptide thatinduces a neutralizing antibody response against all four dengue virusserotypes comprises an amino acid sequence having at least about 90%sequence identity to the amino acid sequence of at least one of SEQ IDNOS:66, 67, 69, 89, 93, and 108-110.

[0203] In another aspect, the invention further provides a recombinantPRM15/truncated E polypeptide that comprises an immunogenic amino acidsequence of the sequence pattern Met Xaa₁ Xaa₂ Xaa₃ Phe Ile Leu Xaa₄ MetLeu Val Xaa₅ Pro Ser Xaa₆ Xaa₇ Met Arg Cys Xaa₈ Gly Xaa₉ Xaa₁₀ Asn Xaa₁₁Asp Phe Val Glu Gly Xaa₁₂ Ser Gly Xaa₁₃ Xaa₁₄ Trp Val Asp Xaa₁₅ Val LeuGlu His Gly Xaa₁₆ Cys Val Thr Thr Met Ala Xaa₁₇ Xaa₁₈ Lys Pro Thr LeuAsp Xaa₁₉ Glu Leu Xaa₂₀ Lys Thr Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Ala Xaa₂₆Leu Arg Xaa₂₇ Xaa₂₈ Cys Ile Glu Ala Xaa₂₉ Xaa₃₀ Xaa₃₁ Asn Xaa₃₂ Thr ThrXaa₃₃ Xaa₃₄ Arg Cys Pro Thr Gln Gly Glu Xaa₃₅ Xaa₃₆ Xaa₃₇ Xaa₃₈ Glu GluGln Asp Xaa₃₉ Xaa₄₀ Xaa₄₁ Xaa₄₂ Cys Xaa₄₃ Xaa₄₄ Xaa₄₅ Xaa₄₆ Val Asp ArgGly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Xaa₄₇ Xaa₄₈ Xaa₄₉ ThrCys Ala Xaa₅₀ Phe Xaa₅₁ Cys Xaa₅₂ Xaa₅₃ Xaa₅₄ Xaa₅₅ Glu Gly Xaa₅₆ Xaa₅₇Val Gln Xaa₅₈ Glu Asn Leu Xaa₅₉ Tyr Xaa₆₀ Xaa₆₁ Xaa₆₂ Xaa₆₃ Thr Xaa₆₄His Xaa₆₅ Gly Xaa₆₆ Xaa₆₇ His Xaa₆₈ Val Gly Asn Xaa₆₉ Thr Xaa₇₀ Xaa₇₁Xaa₇₂ Gly Xaa₇₃ Xaa₇₄ Xaa₇₅ Xaa₇₆ Ile Thr Pro Gln Xaa₇₇ Xaa₇₈ Xaa₇₉Xaa₈₀ Glu Xaa₈₁ Xaa₈₂ Leu Xaa₈₃ Xaa₈₄ Tyr Gly Xaa₈₅ Xaa₈₆ Xaa₈₇ Xaa₈₈Xaa₈₉ Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Xaa₉₀ Xaa₉₁ Xaa₉₂ LeuXaa₉₃ Xaa₉₄ Met Lys Xaa₉₅ Lys Xaa₉₆ Trp Xaa₉₇ Val His Xaa₉₈ Gln TrpXaa₉₉ Xaa₁₀₀ Asp Leu Pro Leu Pro Trp Thr Xaa₁₀₁ Gly Ala Xaa₁₀₂ ThrXaa₁₀₃ Xaa₁₀₄ Xaa₁₀₅ Xaa₁₀₆ Trp Asn Xaa₁₀₇ Lys Glu Xaa₁₀₈ Xaa₁₀₉ Val ThrPhe Lys Xaa₁₁₀ Xaa₁₁₁ His Ala Lys Xaa₁₁₂ Gln Xaa₁₁₃ Val Xaa₁₁₄ Val LeuGly Ser Gln Glu Gly Xaa₁₁₅ Met His Xaa₁₁₆ Ala Leu Xaa₁₁₇ Gly Xaa₁₁₈ ThrGlu Xaa₁₁₉ Xaa₁₂₀ Xaa₁₂₁ Xaa₁₂₂ Xaa₁₂₃ Gly Xaa₁₂₄ Thr Xaa₁₂₅ Xaa₁₂₆ PheXaa₁₂₇ Gly Xaa₁₂₈ Leu Lys Cys Xaa₁₂₉ Xaa₁₃₀ Xaa₁₃₁ Met Xaa₁₃₂ Lys LeuXaa₁₃₃ Xaa₁₃₄ Lys Gly Xaa₁₃₅ Ser Tyr Xaa₁₃₆ Met Cys Thr Gly Xaa₁₃₇ PheXaa₁₃₈ Xaa₁₃₉ Xaa₁₄₀ Lys Glu Xaa₁₄₁ Ala Glu Thr Gln His Gly Thr Xaa₁₄₂Xaa₁₄₃ Xaa₁₄₄ Xaa₁₄₅ Val Xaa₁₄₆ Tyr Xaa₁₄₇ Gly Xaa₁₄₈ Xaa₁₄₉ Xaa₁₅₀ ProCys Lys Ile Pro Xaa₁₅₁ Xaa₁₅₂ Xaa₁₅₃ Xaa₁₅₄ Asp Xaa₁₅₅ Xaa₁₅₆ Xaa₁₅₇Xaa₁₅₈ Xaa₁₅₉ Xaa₁₆₀ Xaa₁₆₁ Gly Arg Leu Ile Thr Xaa₁₆₂ Asn Pro Xaa₁₆₃Val Xaa₁₆₄ Xaa₁₆₅ Lys Xaa₁₆₆ Xaa₁₆₇ Pro Val Asn Ile Glu Xaa₁₆₈ Glu ProPro Phe Gly Xaa₁₆₉ Ser Xaa₁₇₀ Ile Xaa₁₇₁ Xaa₁₇₂ Gly Xaa₁₇₃ Xaa₁₇₄ Xaa₁₇₅Xaa₁₇₆ Xaa₁₇₇ Leu Xaa₁₇₈ Xaa₁₇₉ Xaa₁₈₀ Trp Xaa₁₈₁ Xaa₁₈₂ Lys Gly Ser SerIle Gly Xaa₁₈₃ Met Phe Glu Xaa₁₈₄ Thr Xaa₁₈₅ Arg Gly Ala Xaa₁₈₆ Arg MetAla Ile Leu Gly Xaa₁₈₇ Thr Ala Trp Asp Xaa₁₈₈ Gly Ser Xaa₁₈₉ Xaa₁₉₀Xaa₁₉₁ Xaa₁₉₂ Xaa₁₉₃ Xaa₁₉₄ Xaa₁₉₅ Xaa₁₉₆ Xaa₁₉₇ Xaa₁₉₈ Xaa₁₉₉ Xaa₂₀₀Xaa₂₀₁ Xaa₂₀₂ Xaa₂₀₃ Xaa₂₀₄ Xaa₂₀₅ Xaa₂₀₆ Xaa₂₀₇ Xaa₂₀₈ Xaa₂₀₉ Xaa₂₁₀Xaa₂₁₁ Xaa₂₁₂ Xaa₂₁₃ (SEQ ID NO:51) wherein Xaa at a particular positionrepresents either any or no amino acid residue at that position(typically and preferably, less than twenty of the variable positionsare single residue deletions—i.e., represent no amino acid at theindicated position). Preferably, the polypeptide in this aspecttypically is characterized by the presence of naturally occurring aminoacids, and preferably amino acids that have a hydropathy score above 0.Preferred amino acids for each of the variable positions (as designatedby the subscripted numbers in the sequence pattern) are set forth inTable 3. TABLE 3 X₁: A V T G X₂: V I X₃: I F X₄: L M X₅: A T X₆: Y M X7: A T G X₈: V I X₉: V I T X₁₀: S G X₁₁: R G X₁₂: L V X₁₃: A G X₁₄: A TS X₁₅: L V X₁₆: S G X₁₇: K R Q X₁₈: G N X₁₉: I F X₂₀: L I Q X₂₁: T I EX₂₂: A V X₂₃: K T X₂₄: Q E N X₂₅: L V P X₂₆: L V T X₂₇: K T X₂₈: L YX₂₉: K S X₃₀: L I X₃₁: T S X₃₂: I T X₃₃: A E D X₃₄: T S X₃₅: A P X₃₆: IT Y N X₃₇: L M X₃₈: K V P X₃₉: T Q X₄₀: Q N X₄₁: F Y X₄₂: V I X₄₃: K RX₄₄: H R X₄₅: T S D X₄₆: V F Y M X₄₇: S G X₄₈: L V I X₄₉: V I X₅₀: K MX₅₁: K T Q X₅₂: L V K X₅₃: K T E X₅₄: K P N X₅₅: L I M X₅₆: K N X₅₇: V IX₅₈: H P Y X₅₉: K E X₆₀: T S X₆₁: V I X₆₂: V I X₆₃: V I X₆₄: V P X₆₅: TS X₆₆: E D X₆₇: Q E X₆₈: A Q X₆₉: E D X₇₀: T S G - X₇₁: K E N - X₇₂: H QX₇₃: V K T X₇₄: T I E X₇₅: A V I X₇₆: K T E X₇₇: A S X₇₈: S P X₇₉: T IX₈₀: V T S X₈₁: A I X₈₂: I Q E X₈₃: T P X₈₄: G E D X₈₅: A T X₈₆: L VX₈₇: T G X₈₈: L M X₈₉: E D X₉₀: R E X₉₁: V M X₉₂: V I X₉₃: L M X₉₄: K TX₉₅: K S N X₉₆: A T S X₉₇: L M X₉₈: K R G X₉₉: L F X₁₀₀: L F X₁₀₁: A SX₁₀₂: T S D X₁₀₃: S E X₁₀₄: V T Q E X₁₀₅: V H E P X₁₀₆: T H - X₁₀₇: H RX₁₀₈: L R X₁₀₉: L M X₁₁₀: V T N X₁₁₁: A P X₁₁₂: K R X₁₁₃: E D X₁₁₄: V TX₁₁₅: A T X₁₁₆: T S X₁₁₇: A T X₁₁₈: A T X₁₁₉: V I X₁₂₀: Q D X₁₂₁: T S NM X₁₂₂: S G X₁₂₃: S D X₁₂₄: TN X₁₂₅: L T H X₁₂₆: L I M X₁₂₇: A T X₁₂₈: HR X₁₂₉: K R X₁₃₀: L V X₁₃₁: K R X₁₃₂: E D X₁₃₃: T R Q X₁₃₄: L I X₁₃₅: VM X₁₃₆: V T S X₁₃₇: K S X₁₃₈: K Q X₁₃₉: L I X₁₄₀: V E X₁₄₁: V I X₁₄₂: VI X₁₄₃: L V X₁₄₄: V I X₁₄₅: K R Q X₁₄₆: K Q E X₁₄₇: K E X₁₄₈: T E DX₁₄₉: G D X₁₅₀: A S X₁₅₁: L V F X₁₅₂: S E X₁₅₃: I T S X₁₅₄: Q E M X₁₅₅:L G E X₁₅₆: K Q E X₁₅₇: K G X₁₅₈: K V R X₁₅₉: A T H X₁₆₀: V H Q X₁₆₁: LN X₁₆₂: A V X₁₆₃: A V I X₁₆₄: I T X₁₆₅: K E D X₁₆₆: E D X₁₆₇: K S EX₁₆₈: L A X₁₆₉: E D X₁₇₀: Y N X₁₇₁: V I X₁₇₂: V I X₁₇₃: A V I X₁₇₄: G EX₁₇₅: E P D X₁₇₆: K S G X₁₇₇: A Q X₁₇₈: K T X₁₇₉: L I X₁₈₀: S H N X₁₈₁:F Y X₁₈₂: K R X₁₈₃: K Q X₁₈₄: A T S X₁₈₅: A Y M X₁₈₆: K R X₁₈₇: E DX₁₈₈: L F X₁₈₉: A L V I X₁₉₀: G Y X₁₉₁: T G X₁₉₂: L V - X₁₉₃: L F -X₁₉₄: T N - X₁₉₅: S - X₁₉₆: L V I - X₁₉₇: G - X₁₉₈: K - X₁₉₉: A - MX₂₀₀: L V - X₂₀₁: H - X₂₀₂: Q - X₂₀₃: V I - X₂₀₄: F - X₂₀₅: G - X₂₀₆: AS - X₂₀₇: V I - X₂₀₈: F Y - X₂₀₉: T G - X₂₁₀: A T S - X₂₁₁: V - M X₂₁₂:G F - X₂₁₃: K G -

[0204] As used in Table 3, a dash (-) represents that a single residuedeletion can be preferred at the indicated position in the sequencepattern (i.e., the particular position in the sequence pattern can lackany amino acid residue). Desirably, the polypeptide has an amino acidsequence wherein each of the above-identified variable positions isfilled by one of the preferred residues listed in Table 2 (or a singleresidue deletion, if applicable).

[0205] In a particularly preferred aspect, the invention provides arecombinant PRM15/truncated E polypeptide comprising an immunogenicamino acid sequence of the sequence pattern Met Xaa₁ Xaa₂ Xaa₃ Phe IleLeu Xaa₄ Met Leu Val Xaa₅ Pro Ser Xaa₆ Xaa₇ Met Arg Cys Val Gly Xaa₈ GlyAsn Arg Asp Phe Val Glu Gly Xaa₉ Ser Gly Xaa₁₀ Xaa₁₁ Trp Val Asp Xaa₁₂Val Leu Glu His Gly Xaa₁₃ Cys Val Thr Thr Met Ala Lys Asn Lys Pro ThrLeu Asp Xaa₁₄ Glu Leu Xaa₁₅ Lys Thr Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀ AlaXaa₂₁ Leu Arg Xaa₂₂ Xaa₂₃ Cys Ile Glu Ala Xaa₂₄ Ile Xaa₂₅ Asn Xaa₂₆ ThrThr Xaa₂₇ Xaa₂₈ Arg Cys Pro Thr Gln Gly Glu Xaa₂₉ Xaa₃₀ Leu Xaa₃₁ GluGlu Gln Asp Xaa₃₂ Xaa₃₃ Xaa₃₄ Xaa₃₅ Cys Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉ Val AspArg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Xaa₄₀ Xaa₄₁ ThrCys Ala Lys Phe Xaa₄₂ Cys Xaa₄₃ Xaa₄₄ Xaa₄₅ Xaa₄₆ Glu Gly Xaa₄₇ Xaa₄₈Val Gln Xaa₄₉ Glu Asn Leu Xaa₅₀ Tyr Thr Xaa₅₁ Xaa₅₂ Ile Thr Xaa₅₃ HisXaa₅₄ Gly Xaa₅₅ Xaa₅₆ His Xaa₅₇ Val Gly Asn Asp Thr Xaa₅₈ Xaa₅₉ Xaa₆₀Gly Xaa₆₁ Xaa₆₂ Xaa₆₃ Xaa₆₄ Ile Thr Pro Gln Xaa₆₅ Ser Xaa₆₆ Xaa₆₇ GluAla Xaa₆₈ Leu Xaa₆₉ Xaa₇₀ Tyr Gly Thr Xaa₇₁ Xaa₇₂ Xaa₇₃ Glu Cys Ser ProArg Thr Gly Leu Asp Phe Asn Xaa₇₄ Xaa₇₅ Xaa₇₆ Leu Leu Thr Met Lys Xaa₇₇Lys Xaa₇₈ Trp Xaa₇₉ Val His Xaa₈₀ Gln Trp Phe Xaa₈₁ Asp Leu Pro Leu ProTrp Thr Xaa₈₂ Gly Ala Xaa₈₃ Thr Xaa₈₄ Xaa₈₅ Xaa₈₆ Xaa₈₇ Trp Asn Xaa₈ gLys Glu Xaa₈₉ Xaa₉₀ Val Thr Phe Lys Xaa₉₁ Xaa₉₂ His Ala Lys Xaa₉₃ GlnXaa₉₄ Val Xaa₉₅ Val Leu Gly Ser Gln Glu Gly Ala Met His Xaa₉₆ Ala LeuXaa₉₇ Gly Ala Thr Glu Xaa₉₈ Xaa₉₉ Xaa₁₀₀ Xaa₁₀₁ Xaa₁₀₂ Gly Xaa₁₀₃ Xaa₁₀₄Xaa₁₀₅ Xaa₁₀₆ Phe Xaa₁₀₇ Gly His Leu Lys Cys Xaa₁₀₈ Xaa₁₀₉ Xaa₁₁₀ MetAsp Lys Leu Xaa₁₁₁ Leu Lys Gly Xaa₁₁₂ Ser Tyr Xaa₁₁₃ Met Cys Thr GlyXaa₁₁₄ Phe Xaa₁₁₅ Xaa₁₁₆ Xaa₁₁₇ Lys Glu Xaa₁₁₈ Ala Glu Thr Gln His GlyThr Xaa₁₁₉ Xaa₁₂₀ Xaa₁₂₁ Xaa₁₂₂ Val Xaa₁₂₃ Tyr Xaa₁₂₄ Gly Xaa₁₂₅ Xaa₁₂₆Xaa₁₂₇ Pro Cys Lys Ile Pro Xaa₁₂₈ Xaa₁₂₉ Xaa₁₃₀ Xaa₁₃₁ Asp Xaa₁₃₂ Xaa₁₃₃Xaa₁₃₄ Xaa₁₃₅ Xaa₁₃₆ Xaa₁₃₇ Xaa₁₃₈ Gly Arg Leu Ile Thr Xaa₁₃₉ Asn ProXaa₁₄₀ Val Xaa₁₄₁ Xaa₁₄₂ Lys Xaa₁₄₃ Xaa₁₄₄ Pro Val Asn Ile Glu Xaa₁₄₅Glu Pro Pro Phe Gly Xaa₁₄₆ Ser Xaa₁₄₇ Ile Xaa₁₄₈ Xaa₁₄₉ Gly Xaa₁₅₀Xaa₁₅₁ Xaa₁₅₂ Xaa₁₅₃ Xaa₁₅₄ Leu Xaa₁₅₅ Xaa₁₅₆ Xaa₁₅₇ Trp Xaa₁₅₈ Xaa₁₅₉Lys Gly Ser Ser Ile Gly Xaa₁₆₀ Met Phe Glu Xaa₁₆₁ Thr Xaa₁₆₂ Arg Gly AlaXaa₁₆₃ Arg Met Ala Ile Leu Gly Xaa₁₆₄ Thr Ala Trp Asp Phe Gly Ser Xaa₁₆₅Gly Gly Xaa₁₆₆ Xaa₁₆₇ Thr Ser Xaa₁₆₈ Gly Lys Xaa₁₆₉ Xaa₁₇₀ His GlnXaa₁₇₁ Phe Gly Xaa₁₇₂ Xaa₁₇₃ Tyr Xaa₁₇₄ Xaa₁₇₅ Xaa₁₇₆ Xaa₁₇₇ Xaa₁₇₈ (SEQID NO:50), wherein Xaa represents any amino acid residue (usually anaturally occurring amino acid residue, and more preferably an aminoacid residue with a hydropathy score of above 0) or a single residuedeletion, as described above (typically, less than twenty of thevariable positions are single residue deletions—i.e., represent no aminoacid at the indicated position).

[0206] Preferred amino acids for the each variable positions (asdesignated by the subscripted numbers in the sequence pattern) are setforth in Table 4. TABLE 4 X₁: V T G X₂: V I X₃: I F X₄: L M X₅: A T X₆:Y M X₇: A T G X₈: V I X₉: L V X₁₀: A G X₁₁: A T X₁₂: L V X₁₃: S G X₁₄: IF X₁₅: L I X₁₆: T E X₁₇: A V X₁₈: K T X₁₉: E N X₂₀: V P X₂₁: L V T X₂₂:K T X₂₃: L Y X₂₄: K S X₂₅: T S X₂₆: I T X₂₇: A D X₂₈: T S X₂₉: A P X₃₀:I T Y X₃₁: K V P X₃₂: T Q X₃₃: Q N X₃₄: F Y X₃₅: V I X₃₆: K R X₃₇: H RX₃₈: T D X₃₉: V F Y X₄₀: L V X₄₁: V I X₄₂: K T Q X₄₃: L V K X₄₄: K T EX₄₅: K P N X₄₆: L I M X₄₇: K N X₄₈: V I X₄₉: H P X₅₀: K E X₅₁: V I X₅₂:V I X₅₃: V P X₅₄: T S X₅₅: E D X₅₆: Q E X₅₇: A Q X₅₈: S G - X₅₉: K N -X₆₀: H Q X₆₁: V K X₆₂: T E X₆₃: V I X₆₄: K E X₆₅: A S X₆₆: T I X₆₇: V TX₆₈: I E X₆₉: T P X₇₀: G E X₇₁: L V X₇₂: T G X₇₃: L M X₇₄: R E X₇₅: V MX₇₆: V I X₇₇: K N X₇₈: A T S X₇₉: L M X₈₀: K R X₈₁: L F X₈₂: A S X₈₃: TD X₈₄: S E X₈₅: T E X₈₆: V P X₈₇: T H X₈₈: H R X₈₉: L R X₉₀: L M X₉₁: VT N X₉₂: A P X₉₃: K R X₉₄: E D X₉₅: V T X₉₆: T S X₉₇: A T X₉₈: V I X₉₉:Q D X₁₀₀: S M X₁₀₁: S G X₁₀₂: S D X₁₀₃: T N X₁₀₄: T - X₁₀₅: L T X₁₀₆: LI X₁₀₇: A T X₁₀₈: K R X₁₀₉: L V X₁₁₀: K R X₁₁₁: T Q X₁₁₂: V M X₁₁₃: V TS X₁₁₄: K S X₁₁₅: K Q X₁₁₆: L I X₁₁₇: V E X₁₁₈: V I X₁₁₉: V I X₁₂₀: L VX₁₂₁: V I X₁₂₂: K R Q X₁₂₃: K Q E X₁₂₄: K E X₁₂₅: T E D X₁₂₆: G D X₁₂₇:A S X₁₂₈: L F X₁₂₉: S E X₁₃₀: T I S X₁₃₁: Q E M X₁₃₂: L G E X₁₃₃: K Q EX₁₃₄: K G X₁₃₅: V K R X₁₃₆: A T H X₁₃₇: V H Q X₁₃₈: L N X₁₃₉: A V X₁₄₀:V I X₁₄₁: T I X₁₄₂: K E D X₁₄₃: E D X₁₄₄: K S E X₁₄₅: A L X₁₄₆: E DX₁₄₇: Y N X₁₄₈: V I X₁₄₉: V I X₁₅₀: A V I X₁₅₁: G E X₁₅₂: E P D X₁₅₃: KS G X₁₅₄: A Q X₁₅₅: K T X₁₅₆: L I X₁₅₇: S H N X₁₅₈: F Y X₁₅₉: K R X₁₆₀:K Q X₁₆₁: A T S X₁₆₂: A Y M X₁₆₃: K R X₁₆₄: E D X₁₆₅: L V X₁₆₆: L VX₁₆₇: L F X₁₆₈: L I X₁₆₉: A M X₁₇₀: L V X₁₇₁: V I X₁₇₂: A S X₁₇₃: V IX₁₇₄: T G X₁₇₅: A T X₁₇₆: - M X₁₇₇: F - X₁₇₈: G -

[0207] Preferred amino acids for the each variable positions (asdesignated by the subscripted numbers in the sequence pattern) are setforth in Table 4. Desirably, such polypeptide has an immunogenic aminoacid sequence wherein each of the above-identified variable positions isfilled by one of the residues listed in Table 3 (or a single residuedeletion, if applicable).

[0208] The invention also provides a recombinant truncated E polypeptidethat induces an immune response against at least one dengue virus ofeach of at least two serotypes that is about equal to or greater thanthe immune response induced against the at least one dengue of each ofthe at least two serotypes by a combination of wild-type PRM15/truncatedE polypeptides of the at least two serotypes, wherein each saidwild-type PRM15/truncated E polypeptide is selected from SEQ IDNOS:149-152.

[0209] C15/Full Length prM/Full Length E Polypeptides

[0210] The invention also provides a recombinant or syntheticpolypeptide comprising an amino acid sequence that has at least about70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more amino acid sequence identity to an amino acidsequence of at least one of SEQ ID NOS:139-148, 236-253, 343, and 345.In another aspect, a recombinant or synthetic polypeptide comprising anamino acid sequence that has at least about 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreamino acid sequence identity to an amino acid sequence of at least oneof SEQ ID NOS:139-145, 147-148, 236-253, 343, and 345 is provided. Suchpolypeptides of the invention are typically termed C15/full lengthprM/full length E protein polypeptides (or simply “C15/full prM/full E″polypeptides”).

[0211] Such C15/full length prM/full length E polypeptides induce animmune response in a subject, e.g., mammal against at least one denguevirus of at least one serotype selected from the group of dengue-1,dengue-2, dengue-3, and dengue-4. Further, some such polypeptides inducean immune response in a subject against at least one dengue virus ofeach of at least two, preferably at least three, and more preferably atleast four serotypes selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4. Preferably, the immune response induced againstat least one dengue virus of the at least one serotypes is about equalto or greater than an immune response induced against the at least onedengue virus of the at least one serotype by a corresponding WT C15/fulllength prM/full length E fusion protein of the at least one serotype,wherein the WT C15/full length prM/full length E fusion protein of thesame serotype for comparison is selected from SEQ ID NOS:227-230.

[0212] In one particular aspect, the immune response induced by therecombinant C15/full prM/full E polypeptide induced against at least onedengue virus of each of the four serotypes is about equal to or greaterthan that induced against that at least one dengue virus by any sequenceselected from the group of SEQ ID NOS:227-230.

[0213] Such C15/full prM/full E polypeptides induce production of one ormore antibodies that bind to at least one dengue virus of at least oneserotype. Preferably, such polypeptides induces production of one ormore antibodies that bind to at least one dengue virus of each of atleast two, at least three, or preferably at least four dengue virusserotypes.

[0214] Such polypeptides induce the production of a number of antibodiesthat bind to at least one dengue virus of at least one serotype that isabout equal to or greater than the number of antibodies that bind to theat least one dengue virus of the at least one serotype induced by a WTC15/full length prM/full length E fusion protein of the at least onedengue virus of the at least one serotype, wherein the WT C15/fulllength prM/full length E fusion protein of each particular serotype isselected for comparison from SEQ ID NOS:227-230.

[0215] In one aspect, a recombinant C15/full length prM/full length Epolypeptide induces production of a number of antibodies that bind to atleast one dengue virus of each of the at least two or at least threeserotypes, wherein the number is about equal to or greater than thenumber of antibodies that bind to the at least one dengue virus of eachof the at least two or at least three serotypes that are induced by awild-type C15/full length prM/full length E polypeptide corresponding toeach of the at least two or three serotypes selected from SEQ IDNOS:227-230, respectively. In one particular embodiment, a C15/fullprM/full E polypeptide induces production of a number of antibodies thatbind to at least one dengue virus of each of dengue-1, dengue-2,dengue-3, and dengue-4 that is about equal to or greater the number ofantibodies that bind to the at least one dengue virus of each of thosefour serotypes that are induced by any one SEQ ID NOS:227-230,respectively.

[0216] Such recombinant wild-type C15/full length prM/full length Epolypeptides induce the production of antibodies that bind morespecifically to at least one particular dengue virus of at least oneparticular serotype than is induced by a corresponding wild-typeC15/full length prM/full length E polypeptide from the dengue virus ofthe same one serotype, wherein the wild-type C15/full length prM/fulllength E polypeptide is selected from SEQ ID NOS:227-230.

[0217] Another characteristic of a recombinant C15/full length prM/fulllength E polypeptide of the invention is the ability to induce theproduction of a titer of neutralizing antibodies against at the leastone dengue virus of the at least one serotype. In one aspect, thepolypeptide produces a titer of neutralizing antibodies against at leastone dengue virus of each of at least two, at least three, or at leastfour serotypes. Some such polypeptides induce at least one neutralizingantibody response in a subject to or against at least one dengue virusof each of at least two, three or four serotypes without an occurrenceof antibody-dependent enhancement (ADE) upon contact of the subject withthe at least dengue virus of each of the at least two, three, or fourserotypes, respectively.

[0218] Some such polypeptides produce a titer of neutralizing antibodiesin a subject against at least one dengue virus of at least one serotypethat is about equal to or greater than a titer of neutralizingantibodies produced in the subject against the at least one dengue virusof the at least one serotype by a WT C15/full length prM/full length Efusion protein of the at least one dengue virus of the at least oneserotype. The WT C15/full length prM/full length E fusion protein can beselected from SEQ ID NOS:227-230. Further, some polypeptides produce atiter of neutralizing antibodies against at least one dengue virus ofeach of at least two, three, or four serotypes that is about equal to orgreater than a titer of neutralizing antibodies produced against the atleast one dengue virus of each of at least two, three, or four serotypesby a WT C15/full length prM/full length E fusion protein of the at leastone dengue virus of each of the at least two, three, or four serotypes,respectively, wherein each WT C15/full length prM/full length Epolypeptide is selected from SEQ ID NOS:227-230.

[0219] In one aspect, polypeptides having these immunogenic andimmune-stimulating properties comprise an amino acid sequence that hasat least about 95% amino acid sequence identity to the amino acidsequence of SEQ ID NO:140 or SEQ ID NO:141. In one particular aspect,such a polypeptide comprises SEQ ID NO:141.

[0220] Some such C15/full length prM/full length E polypeptides of theinvention induce an immune response against at least one dengue virus ofeach of at least two serotypes that is about equal to or greater thanthe immune response induced against any of the at least one dengue virusof each of the at least two serotypes by a combination of WT C15/fulllength prM/full length E polypeptides of each of the at least twoserotypes. WT C15/full length prM/full length E polypeptides can beselected from the group of SEQ ID NOS:227-230.

[0221] Recombinant C15/full length prM/full length E polypeptidesexhibit additional biological properties that can be favorable forinducing, promoting, modulating, and/or enhancing an immune response,such as, e.g., the ability to form immunogenic viruses or virus-likeparticles in cells of a subject, including, e.g., mammalian cells. Theinvention provides a population of such recombinant polypeptides,wherein such polypeptides are capable of assembling with one another(and with other polypeptides and nucleic acids, as desired) to form oneor more VLPs or viruses.

[0222] The invention also provides an antigenic or immunogenic fragmentof any such polypeptide of the invention. The antigenic or immunogenicfragment of the polypeptide may comprise an amino acid sequence of about10, about 20, about 30, or about 50 amino acids that comprises at leastone T cell epitope not present in corresponding wild-type dengue virusC15, prM and E protein amino acid sequences, wherein the novel epitopeis derived from one of the novel immunogenic amino acid sequencesdisclosed herein. Extension of the polypeptide by, e.g., inclusion ofadditional amino acid residue(s) and/or polypeptide or peptide segment(e.g., a signal peptide sequence or C-terminal E protein sequence,N-terminal prM protein sequence, or C15/N-terminal prM protein sequence)increases the length of the polypeptide. Common and preferable sizes forsuch polypeptides are further discussed elsewhere herein.

[0223] C Terminal E Protein Fragment Polypeptides

[0224] In one aspect of the invention, recombinant immunogenic orantigenic truncated E polypeptides, PRM15/truncated E polypeptides, andC15/full length prM/truncated E polypeptides of the invention mayfurther comprise an additional amino acid sequence that is similar,substantially similar, or identical to an amino sequence segment orfragment (e.g., hydrophobic amino acid sequence) of the C-terminustransmembrane domain of a wild-type E protein of a flavivirus or arecombinant E protein of a flavivirus (such as, preferably, a denguevirus or yellow fever virus). Such amino acid sequence, which may betermed a C terminal amino acid fragment of a (flavivirus) E protein, Cterminal E protein fragment (or simply “rest of envelope” or “rest ofenv” sequence), is typically about 20 to about 70 amino acid residues inlength, more typically about 40 to about 65 amino acid residues inlength, and often about 40 to about 65 amino acid residues in length.Such an amino acid fragment may comprise the amino acid sequencecorresponding to the “stem-anchor region” of an E protein, which wouldtypically anchor the remainder of the E protein (e.g., the truncated Eprotein portion) to the cell membrane when such E protein wasincorporated into a virus.

[0225] Some such C terminal E protein fragments each comprise an aminoacid sequence that has at least about 45%, desirably at least about 50%or 55% (e.g., about 60-99%), favorably at least about 60% or 65%, morefavorably at least about 70% or 75%, advantageously at least about 80%or 85%, and preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% or more amino acid sequence identity to at least oneamino acid sequence of the group of SEQ ID NOS:127-136.

[0226] Each such C terminal E protein fragment sequence is positionednear to (e.g., is within about 20 amino acids of) or fused to any of thefollowing: 1) the C terminal amino acid of a recombinant truncatedenvelope (tE) protein polypeptide of the invention (e.g., SEQ IDNOS:1-49 and 154-155); 2) the C terminal amino acid of a recombinantPRM15/tE polypeptide of the invention (e.g., SEQ ID NOS:65-116); 3) theC terminal amino acid of a recombinant full length prM/tE fusion proteinof the invention; or 4) the C terminal amino acid of a recombinantC15/full length prM/tE protein polypeptide of the invention as describeabove. Such C terminal E protein fragment serves to extend the length ofthe truncated E polypeptide to a length that is about equal to orsubstantially equivalent to the length of a full length E polypeptide ofa wildtype flavivirus, preferably a dengue virus of a particularserotype. Thus, a recombinant truncated E polypeptide, PRM15/truncated Epolypeptide, and C15/full length prM/truncated E polypeptide of theinvention that further comprises such a C terminal E polypeptide aretypically referred to as a recombinant full length E polypeptide,PRM15/full length E polypeptide, and C15/full length prM/full length Epolypeptide, respectively.

[0227] In one aspect, the amino acid sequence of such C terminal aminoacid fragment of a (flavivirus) E protein comprises, or typicallyconsists essentially of, an amino acid sequence according to thesequence pattern: Gly Val Ser Trp Xaa₁ Xaa₂ Xaa₃ Ile Xaa₄ Ile Gly Xaa₅Xaa₆ Xaa₇ Xaa₈ Trp Xaa₉ Gly Xaa₁₀ Asn Ser Xaa₁₁ Xaa₁₂ Thr Ser Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀ Gly Xaa₂₁ Xaa₂₂ Thr Leu Xaa₂₃Leu Gly Xaa₂₄ Xaa₂₅ Val Xaa₂₆ Ala, wherein Xaa represents any amino acidresidue (see SEQ ID NO:137). Preferred amino acid residues for thevariable positions in this sequence pattern are provided in Table 5.TABLE 5 X₁: M I T X₂: V M X₃: K R X₄: L G X₅: V I F X₆: I L X₇: I L VX₈: T L X₉: I L X₁₀: M L T X₁₁: K R X₁₂: S N X₁₃: L M X₁₄: S A X₁₅: V MF X₁₆: S T X₁₇: L C X₁₈: V I X₁₉: L A X₂₀: V I X₂₁: V M I G X₂₂: V IX₂₃: Y F X₂₄: A V F X₂₅: M V T X₂₆: Q H

[0228] Desirably, each one of the variable positions in this sequencepattern is filled by one of the preferred residues provided in Table 5.

[0229] The invention also includes polynucleotides encoding all suchrecombinant polypeptides as described herein and below. Suchpolypeptides and polypeptide-encoding nucleotides are useful in methodsof the invention described throughout, including, e.g., but not limitedto, prophylactic and/or therapeutic methods of treatment to induce,modulate, enhance, and/or promote an immune response(s) to at least onedengue virus of at least one flavivirus serotype in a mammal, and/ormethods of detecting or diagnosing the presence of antibodies in asample that bind to one or more dengue viruses of one or more serotypes.

[0230] N Terminal C15/Truncated prM Polypeptides

[0231] Recombinant PRM15/tE polypeptides and PRM15/full E polypeptidesmay further comprise an additional amino acid sequence that has at leastabout 50%, desirably at least about 60% (e.g., about 65% to about 100%),favorably at least about 70%, preferably at least about 80%, and morepreferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% or more amino acid sequence identity to an amino acid sequence ofat least one of the group of SEQ ID NOS:117-126. In one embodiment, suchan amino acid sequence comprises at least the two following segments:(1) the last 15 amino acid residues of recombinant or wild-type capsid(C) protein of a flavivirus (e.g., preferably dengue virus), as measuredin reverse sequence order from the C terminus of the capsid proteinsequence and (2) all of the amino acid residues of a recombinant orwild-type prM protein of a flavivirus (e.g., preferably dengue virus),as measured in sequence order from the N terminus of the prM proteinsequence, except the last 15 amino acid of the C terminus of the prMprotein. Typically, segment (1) and segment (2) are attached or fused,and segment (1) precedes segment (2), positioned at the N terminus. Suchan amino acid sequence is typically termed an N terminal amino acidfragment sequence of C15/prM or an N terminal C15/truncated prMpolypeptide (or “rest of C15/PRM,” since it includes 15 residues fromthe C protein and the remaining residues of the prM protein, but for the15 C terminal residues). Such an N terminal C15/truncated prMpolypeptide is usually at least about 150, 160, 165, 170, 175, or 180amino acid residues in length. Some such polypeptide fragments are atleast about 165 to about 175 amino acids in length. Some suchpolypeptide fragments are at least about 167 to about 171 amino acids inlength.

[0232] An N terminal C15/truncated prM polypeptide is positioned near toor at (e.g., is within about 20 amino acids of), or is fused directlyto, the N-terminus of the signal peptide sequence or immunogenic aminoacid sequence (e.g., an immunogenic truncated E polypeptide sequence oran immunogenic full length E polypeptide sequence) of an immunogenicpolypeptide of the invention, depending on the position (and presence)of the signal peptide sequence in the polypeptide. For example, in oneformat, an N terminal C15/truncated prM polypeptide is positioned nearor at or is fused directly to the N terminus of a recombinant PRM15/tEdengue virus polypeptide of the invention (e.g., SEQ ID NOS:65-116) orthe N terminus of a recombinant PRM15/full length E polypeptide of theinvention (SEQ ID NOS:139-148, 236-253). In another aspect, the Nterminal C15/truncated prM polypeptide is positioned near or at or isfused directly to the N terminus of a recombinant truncated E denguevirus polypeptide of the invention (e.g., SEQ ID NOS:1-49 and 153-155)or the N terminus of a recombinant full length E polypeptide of theinvention.

[0233] In another aspect, a recombinant PRM15/tE polypeptide orPRM15/full E polypeptide further comprises an N terminal C15/truncatedprM polypeptide positioned near to or at or fused directly to theN-terminus of the PRM15/tE polypeptide or PRM15/full E polypeptide,respectively, wherein the N terminal C15/truncated prM polypeptidecomprises an amino acid sequence according to the sequence pattern setforth in SEQ ID NO:138, which comprises: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Pro Xaa₁₂ Xaa₁₃ Xaa₁₄ Ala Phe Xaa₁₅ Leu Xaa₁₆Xaa₁₇ Arg Xaa₁₈ Gly Glu Pro Xaa₁₉ Xaa₂₀ Ile Val Xaa₂₁ Xaa₂₂ Xaa₂₃ GluXaa₂₄ Gly Xaa₂₅ Xaa₂₆ Leu Leu Phe Lys Thr Xaa₂₇ Xaa₂₈ Gly Xaa₂₉ AsnXaa₃₀ Cys Thr Leu Xaa₃₁ Ala Xaa₃₂ Asp Leu Gly Glu Xaa₃₃ Cys Xaa₃₄ AspThr Xaa₃₅ Thr Tyr Lys Cys Pro Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉ Xaa₄₀ Glu ProXaa₄₁ Asp Xaa₄₂ Asp Cys Trp Cys Asn Xaa₄₃ Thr Xaa₄₄ Xaa₄₅ Trp Val Xaa₄₆Tyr Gly Thr Cys Xaa₄₇ Xaa₄₈ Xaa₄₉ Gly Glu Xaa₅₀ Arg Arg Xaa₅₁ Lys ArgSer Val Ala Leu Xaa₅₂ Pro His Xaa₅₃ Gly Xaa₅₄ Gly Leu Xaa₅₅ Thr ArgXaa₅₆ Xaa₅₇ Thr Trp Met Ser Xaa₅₈ Glu Gly Ala Trp Xaa₅₉ Xaa₆₀ Xaa₆₁Xaa₆₂ Xaa₆₃ Xaa₆₄ Glu Xaa₆₅ Trp Xaa₆₆ Leu Arg Xaa₆₇ Pro Xaa₆₈ Phe Xaa₆₉Xaa₇₀ Xaa₇₁ Ala Xaa₇₂ Xaa₇₃ Xaa₇₄ Ala Xaa₇₅ Xaa₇₆ Ile Gly Xaa₇₇ Xaa₇₈Xaa₇₉ Xaa₈₀ Gln Xaa₈₁, where Xaa represents any amino acid residue.Preferred amino acid residues for the variable (Xaa) positions for sucha N terminal C15/truncated prM polypeptide sequence are provided inTable 6. TABLE 6 X₁: M R X₂: R K X₃: S T X₄: V S T A X₅: T L G I X₆: M CV T X₇: I L X₈: L M I X₉: M C X₁₀: L M X₁₁: L I X₁₂: T A X₁₃: A T V X₁₄:L M X₁₅: H S X₁₆: T S X₁₇: T S X₁₈: G D N X₁₉: T L R H X₂₀: L M X₂₁: S AG X₂₂: K R X₂₃: Q H N X₂₄: R K X₂₅: K R X₂₆: S P X₂₇: S T A E X₂₈: A E SD X₂₉: V I X₃₀: M K X₃₁: I M X₃₂: M I X₃₃: L M X₃₄: E D X₃₅: M V I X₃₆:R L H X₃₇: M L I X₃₈: T R V X₃₉: E Q N X₄₀: A N V T X₄₁: D E X₄₂: V IX₄₃: A L S X₄₄: D S X₄₅: T A X₄₆: T M X₄₇: S N T X₄₈: Q T X₄₉: T A SX₅₀: H R X₅₁: D E X₅₂: D A T V X₅₃: V S X₅₄: L M X₅₅: E D X₅₆: T A X₅₇:E Q X₅₈: S A X₅₉: K R X₆₀: H Q X₆₁: I V A X₆₂: Q E X₆₃: K R X₆₄: V IX₆₅: T S X₆₆: A I X₆₇: H N X₆₈: G R X₆₉: T I A X₇₀: V I L X₇₁: I L MX₇₂: L A G X₇₃: F I X₇₄: L M X₇₅: H Y X₇₆: A Y T M X₇₇: T Q X₇₈: T SX₇₉: I L H G X₈₀: T F I X₈₁: K R

[0234] Desirably, each one of the variable positions of theabove-described sequence pattern is filled by one of the above-listedamino acid residues.

[0235] In some aspects, a recombinant PRM15/tE polypeptide of theinvention (e.g., such as a polypeptide having at least about 85%identity to a polypeptide sequence selected from any of SEQ ID NOS65-116) further comprises an N terminal C15/truncated prM polypeptidecomprising a sequence selected from SEQ ID NOS:117-126 and/or a Cterminal E protein fragment polypeptide comprising a sequence selectedfrom SEQ ID NOS:127-136.

[0236] In other aspects, a polypeptide of the invention, such as, e.g.,a recombinant truncated E polypeptide, PRM15/truncated E polypeptide, orC15/full prM/truncated E polypeptide, desirably comprises a C terminal Eprotein fragment comprising an amino acid sequence that has less than100% sequence identity (e.g., about 99%, 98%, 97%, 96%, 95%, 94%, 90%)with a C terminal E protein fragment sequence of a wild-type denguevirus E protein of one of the four serotypes (e.g., SEQ ID NOS:127-130).A polypeptide of the invention, such as, e.g., a recombinantPRM15/truncated E polypeptide or PRM15/full E polypeptide also oralternatively can comprise an N terminal C15/truncated prM polypeptidethat has less than 100% amino acid sequence identity (e.g., about 99%,98%, 97%, 96%, 95%, 94%, 90%) with an N terminal C15/truncated prMpolypeptide sequence of a wild-type dengue virus prM protein (e.g., SEQID NOS:117-120).

[0237] In some aspects, a C15/full prM/full E polypeptide, whichincludes a C terminal E protein fragment that has less than 100%sequence identity with the sequence of a C terminal E protein of a WTdengue virus E protein and a C15/truncated prM polypeptide that has lessthan 100% sequence identity with the sequence of an N terminalC15/truncated prM polypeptide of a WT dengue virus, comprises apolypeptide sequence that has at least about 65%, at least about 70%,typically at least about 75% or at least about 80%, preferably at leastabout 85% (including e.g., at least about 85 to about 99.5%), and morepreferably at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% or more amino acid sequence identity to at leastone of SEQ ID NOS:139-148, 236-253, 343, and 345. More preferably, sucha polypeptide comprises a polypeptide sequence selected from (or havingat least about 80%, about 85%, about 88%, about 90%, about 95%, 97%,about 98%, or about 99% identity with) SEQ ID NOS:139-148, 236-253, 343,and 345. Polypeptides that have at least about 80%, more preferably atleast about 85%, and even more preferably at least about 90% (e.g.,about 90%, 92%, 93%, 94%, or 95%) identity with SEQ ID NO:140 or SEQ IDNO:141 are a particular aspect of this invention. Preferred polypeptidescomprise (or at least have about 96%, about 97%, about 98%, about 99%,or more identity with) SEQ ID NO:141.

[0238] The invention also provides for the use of a novel C terminal Eprotein fragment polypeptide of the invention (e.g., SEQ ID NOS:131-136)and C15/truncated prM polypeptide of the invention (e.g., SEQ IDNOS:121-126) independently of the inclusion of either in any of theabove-described polypeptides. For example, polypeptides comprising orconsisting essentially of at least one such novel C terminal E proteinfragment polypeptide and/or at least one such novel C15/truncated prMpolypeptide can be used to induce or promote an immune response to aflavivirus, such as a dengue, virus in a mammalian host; can be used inmethods of diagnosis or detecting the presence of antibodies that bindto one or more dengue viruses of a biological sample; and/or can be usedin the formation of dengue virus immunogens or antigens. The use of thenucleic acid sequences encoding these C terminal E protein fragmentpolypeptides and C15/truncated prM polypeptides, alone or in combinationwith other nucleic acid sequences, including those encoding PRM/tEpolypeptides of the invention, also is provided.

[0239] The inventors also contemplate the use of novel PRM15-homologs(also referred to as “prM15 homologs”) of the invention (e.g., SEQ IDNOS:56-64), for example, in the formation of dengue virus immunogens orantigens, as signal peptide sequences for truncated dengue virus Eprotein antigens (e.g., such as SEQ ID NOS:1-49 and 153-155) or fulllength dengue virus E protein antigens), or as signal peptides for otherviral polypeptides, such as flavivirus truncated E proteins or fulllength E proteins, or non-viral polypeptides. The use of the nucleicacid sequences encoding these signal peptides (e.g., portions of thepolynucleotides described herein which code such amino acid sequences),alone or in combination with other nucleic acid sequences, such as,e.g., recombinant truncated E polypeptides of the invention, is alsoprovided. In one aspect, e.g., the use of such a signal peptide nucleicacid sequence with an immunogenic nucleic acid (e.g., nucleic acidencoding any of SEQ-ID NOS:1-49 and 153-155) in a DNA vaccine iscontemplated.

[0240] A immunogenic polypeptide comprising or consisting essentially ofan amino acid sequence that is substantially identical (e.g., having atleast about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity) to anamino acid sequence selected from the group of SEQ ID NOS:1-49 and153-155 is at least about 400 to about 500 amino acids in length (moretypically at least about 440 to about 460 amino acids in length). Thelower size limit for the immunogenic amino acid sequence, and,accordingly, the polypeptide itself, typically is only dictated by thedesired use of the polypeptide. In aspects where the immunogenicpolypeptide is used to promote an immune response to a dengue virus, thesize of the polypeptide can be similar to that of at least a truncateddengue virus envelope protein (e.g., about 440 amino acids).Alternatively, the truncated E polypeptide can be extended to a fulllength E protein polypeptide (having an amino acid sequence about atleast as long as a WT dengue virus E protein or WT flavivirus E protein)as described herein. Alternatively, in methods wherein the polypeptideis used to induce an immune response, the polypeptide comprises aPRM15/truncated E polypeptide (e.g., about 435-465 amino acids, or a C15signal sequence/full length prM/full length E polypeptide (e.g., about650-680 amino acids).

[0241] Viruses and Virus-Like Particles

[0242] The invention also provides recombinant or synthetic viruses andvirus-like particles (VLPs) comprising one or more of the polypeptides,nucleic acids, or vectors of the invention. Such viruses and VLPs, whichmay be attenuated, are useful in methods of inducing, modulating,enhancing or promoting an immune response to at least one flavivirus,preferably dengue virus, of at least one serotype as described herein.Such viruses and VLPs are useful in therapeutic and/or prophylacticmethods to treat flaviviral infection (e.g., infection by one or moredengue viruses) or protect against infection by a flavivirus (e.g.,infection by one or more dengue viruses). Such viruses and VLPs areuseful in vaccines to safeguard against dengue viral infection and/orADE.

[0243] In one aspect, the invention provides a virus comprising (a) anucleic acid comprising a nucleotide sequence that has at least about90%, 95%, or 100% sequence identity to a nucleotide sequence selectedfrom any of SEQ ID NOS:156-218, 235, 254-271, 285-330, 342, and 344,;and/or (b) a polypeptide comprising an amino acid sequence that has atleast about 90%, 95%, or 100% sequence identity to a sequence selectedfrom any of SEQ ID NOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and345.

[0244] Also provided is a chimeric virus comprising: (a) a nucleic acidcomprising a nucleotide sequence that has at least about 90, 95, or 100%sequence identity to a sequence selected from any of SEQ ID NOS:156-218,235, 254-271, 285-330, 342, and 344, and at least one additional nucleicacid from a genome of another virus, including a flavivirus oradenovirus. The flavivirus may be a dengue virus (e.g., DEN-1, DEN-2,DEN-3, DEN-4) or yellow fever virus, Japanese encephalitis virus; Equineencephalitis virus; West Nile virus); and/or (b) a polypeptidecomprising an amino acid sequence having at least about 90%, 95%, or100% sequence identity to a sequence selected from any of SEQ IDNOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and 345 and at leastone additional amino acid comprising a structural or non-structuralpolypeptide of a flavivirus or a fragment of a structural ornon-structural polypeptide of a flavivirus, wherein said flavivirus is adengue virus (e.g., DEN-1, DEN-2, DEN-3, or DEN-4) or is not a denguevirus (e.g., yellow fever virus, Japanese encephalitis virus; Equineencephalitis virus; or West Nile virus).

[0245] In another aspect, the invention provides a method of inducing animmune response in a host against a first flavivirus comprising: (a)providing a nucleic acid comprising a nucleotide sequence that has atleast about 90%, 95%, or 100% sequence identity to a sequence selectedfrom any of SEQ ID NOS:156-218, 235, 254-271, 285-330, 342, and 344,wherein said nucleic acid comprises a DNA sequence; (b) generatinginfectious RNA transcripts from the DNA sequence; (c) introducing theRNA transcripts into a cell; (d) expressing the RNA transcripts in thecell to produce virus; (e) harvesting the virus from said cell; and (g)inoculating the host with virus.

[0246] As used herein, the term “virus” includes not only complete virusparticles, but also virus-like particles (VLPs) that include one or morepolypeptides of the invention. A desirable feature of polypeptidescomprising one of the above-described C15/full length prM/full length Epolypeptides, and full length prM/truncated E polypeptides is theformation of virus-like particles (VLPs) in a mammalian host by apopulation of the polypeptides. VLPs lack the viral components that arerequired for virus replication and thus represent a highly attenuatedform of a virus. A VLP of the invention can display a polypeptide (e.g.,recombinant antigen) that is protective against one or more flavivirusserotypes, preferably one or more dengue virus serotypes. VLPs candisplay more than one type of polypeptide (e.g., recombinant antigen);e.g., a VLP can display and thus are useful as a polyvalent vaccinewhere antigens that are protective. In some embodiments, the methods ofthe invention are used to obtain VLPs that have desired characteristicsor properties as described herein, including e.g., those relating toenhanced cross-protection against and/or cross-reactivity with at leasttwo flavivirus serotypes (preferably at least two dengue virusserotypes), secretion, and/or expression. Viral proteins from severalviruses are known to form VLPs, including human papillomavirus, HIV(Kang et al., Biol. Chem. 380: 353-64 (1999)), Semliki-Forest virus(Notka et al., Biol. Chem. 380: 341-52 (1999)), human polyomavirus(Goldmann et al., J Virol. 73: 4465-9 (1999)), rotavirus (Jiang et al.,Vaccine 17: 1005-13 (1999)), parvovirus (Casal, Biotechnology andApplied Biochemistry, Vol 29, Part 2, pp 141-150 (1999)), canineparvovirus (Hurtado et al., J. Virol 70: 5422-9 (1996)), and hepatitis Evirus (Li et al., J Virol. 71: 7207-13 (1997)).

[0247] The formation of such VLPs can be detected by any suitabletechnique. Examples of suitable techniques known in the art fordetection of VLPs in a medium include, e.g., electron microscopytechniques, dynamic light scattering (DLS), selective chromatographicseparation (e.g., ion exchange, hydrophobic interaction, and/or sizeexclusion chromatographic separation of the VLPs) and density gradientcentrifugation.

[0248] In another aspect, the invention also provides modified, mutant,synthetic or recombinant dengue viruses. Accordingly, the inventionprovides a modified (e.g., mutant, synthetic, or recombinant) denguevirus that comprises at least one recombinant dengue virus nucleic acidor polypeptide of the invention described herein. In one embodiment, theinvention provides a modified or recombinant dengue virus produced byexpression or translation of a recombinant nucleic acid of the inventionin a population of a subject's cells, e.g., mammalian cells, including,e.g., mouse, primate, and/or human cells. In another embodiment, theinvention provides a modified or recombinant dengue virus that comprisesat least one recombinant DNA nucleotide sequence of the inventiondescribed herein. In yet another embodiment, the invention provides amodified or recombinant dengue virus produced by expression ortranslation of an RNA nucleic acid in a population of cells, e.g.,mammalian cells, the RNA nucleic acid comprising an RNA nucleic acidsequence that comprises a modified or recombinant DNA nucleic acidsequence of the invention, wherein each thymine residue of such DNAnucleic acid sequence is replaced by a uracil residue; the inventionalso includes a complementary sequence of each said RNA nucleic acidsequence. In another preferred embodiment, the invention provides amodified or recombinant dengue virus comprising an RNA nucleotidesequence, said RNA nucleotide sequence comprising the isolated orrecombinant DNA nucleic acid sequence of any of SEQ ID NOS:156-218, 235,254-271, 285-330, 342, and 344, wherein each thymine residue in eachsaid DNA nucleotide sequence is replaced by a uracil residue; an RNAsequence that is complementary to each said RNA nucleotide sequence isalso provided.

[0249] Properties and Characteristics

[0250] Recombinant, synthetic, mutant, and/or isolated polypeptides ofthe invention exhibit a variety of properties and characteristics andare useful in a variety of contexts. In one aspect, such polypeptidesare useful in methods of detecting or diagnosing of anti-flaviviralantibodies, especially anti-dengue virus antibodies in a biologicalsample, as described in greater detail below. In another aspect, acharacteristic of the recombinant, synthetic, and/or isolatedpolypeptides of the invention is the promotion of an immune response toat least a portion of a dengue virus (e.g., a dengue virus antigen, acollection of dengue virus antigens, a fragment of a dengue virus, adengue virus VLP, or an inactivated, attenuated, or virulent denguevirus of one, two, three, or even four serotypes) in an animal orpopulation of animal cells, and preferably in a mammal, even morepreferably in a human. “Promotion” encompasses any detectable increase,including induction of an immune response and increase of an alreadyexisting immune response. The polypeptides of the invention can induce acytotoxic (or other T-cell) immune response, a humoral(antibody-mediated) immune response, or (most desirably) both, in such ahost. The polypeptide can promote the production of an antibody thatbinds to at least a portion of a dengue virus (e.g., a particular denguevirus antigen), in a subject, such as a mammal. Desirably, thepolypeptide promotes an anamnestic antibody response to a dengue virus(which can be determined by known IgG/IgM kinetics analysis techniques),and the polypeptide induces or promotes a “solid” (anamnestic andnon-viremic) antibody response to a dengue virus.

[0251] More particular characteristics of immune responses attendant theadministration or expression of one or more polypeptides of theinvention to a subject host, such as a mammal, include the priming andstimulation of CD4+ and CD8+ lymphocytes, particularly CD8+ lymphocytes,the promotion of host cell production of anti-dengue virus IgM and/orIgG antibodies, T cell activation and cytokine release (including, butnot limited to, e.g., release of one or more tumor necrosis factors(TNF) (e.g., TNF-alpha), the production of one or more interleukins (IL)(e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12), the productionof one or more interferons (IFN) (e.g., IFN-gamma, IFN-alpha, IFN-beta),TGF from T cells), complement activation, platelet activation, enhancedand/or decreased Th1 responses, enhanced and/or decreased Th2 responses,and humoral immunological memory.

[0252] An important characteristic of polypeptides of the invention isthe promotion of an immune response to at least one dengue virus of oneor more, preferably multiple, serotypes in a subject. For example, theinvention provides polypeptides comprising an immunogenic amino acidsequence that induces production of one or more antibodies that bind atleast one dengue virus of each of at least two virus serotypes inanimals, such as a mammal.

[0253] More preferred polypeptides of the invention promote an immuneresponse to one or more dengue virus variants of each of at least threevirus serotypes of in an animal, e.g., a mammal. For example, aparticular polypeptide of the invention promote the production of one ormore antibodies that bind at least one dengue virus of each of at leastthree virus serotypes in a mammal when an antigenic or immunogenicquantity of such a polypeptide is expressed in, administered to, ordelivered to the mammal.

[0254] An advantage of polypeptides of the invention is the ability toinduce an immune response to at least a portion (preferably to multipleportions—e.g., multiple epitopes) of at least one dengue virus of atleast one serotype in vivo and ex vivo (in contrast to polypeptides thatonly induce such an immune response in cell culture). Some polypeptidesof the invention induce production of one or more antibodies (anantibody response) to antigens (including dengue virus Ags) of all fourknown dengue virus serotypes in a subject (e.g., including, but notlimited to SEQ ID NOS:2, 3, 5, 25, 29, 66, 67, 69, 89, 93, 44-46,108-110, 140, and 141).

[0255] An advantage of PRM15/tE polypeptides of the invention is theability to induce or promote an immune response to one or more dengueviruses of one or more serotypes that is about equal to or even greaterthan the immune response induced or promoted against such one or dengueviruses of one or more serotypes, respectively, by a PRM15/tEpolypeptide comprising a wild-type dengue virus PRM15/tE polypeptide(e.g., SEQ ID NOS:149-152). Preferably, the PRM15/tE polypeptide iscapable of inducing or promoting an immune response against at least onedengue virus of at least one serotype that is at least equal to orgreater than that induced or promoted against the at least one denguevirus of the at least one serotype by a wild-type dengue virus PRM15/tEantigenic polypeptide of any virus serotype (e.g., more than any of SEQID NOS:149-152). Preferably, the polypeptide of the invention induces animmune response in a subject to at least one dengue virus of at leasttwo, more preferably at least three, even more preferably at least four,virus serotypes, in a mammalian cell that is equal to or greater thanthe immune response induced against said at least one dengue virus of atleast two, at least three, at least four virus serotypes in a subject bywild-type dengue virus PRM15/tE polypeptide of the respective serotype.

[0256] Moreover, some such PRM15/tE polypeptides also or alternativelycan induce or promote an immune response in vivo or ex vivo against atleast one dengue virus of all four serotypes in a subject that is atleast about equal to or greater than the immune response induced orpromoted against at least one dengue virus of all four serotypes by thecombination of wild-type dengue virus PRM15/tE antigenic polypeptides ofall four serotypes (e.g., equal to or greater than the combination ofSEQ ID NOS:149-152).

[0257] An advantage of C15/full prM/full E polypeptides of the inventionis the ability to induce or promote an immune response to at least onedengue virus of at least one serotype that is about equal to or evengreater than the immune response induced or promoted against at leastone dengue virus of the at least one serotype by a wild-type denguevirus C15/full prM/full E polypeptide (e.g., SEQ ID NOS:227-230).Preferably, a C15/full prM/full E polypeptide induces an immune responsein a subject to at least one dengue virus of at least two, morepreferably at least three, even more preferably at least four, virusserotypes, in a subject that is equal to or greater than the immuneresponse induced serotypes in the subject against said at least onedengue virus of at least two, at least three, at least four virus cellby wild-type dengue virus C15/full prM/full E polypeptide of therespective serotype.

[0258] In one aspect, a C15/full prM/full E is capable of inducing orpromoting an immune response against at least one dengue virus of atleast one serotype that is greater than that induced or promoted againstthe respective dengue virus serotype(s) by a C15/full prM/full Eantigenic polypeptide of any serotype (e.g., >than any of SEQ IDNOS:227-230).

[0259] Moreover, some such C15/full prM/full E PRM15/tE polypeptidesalso or alternatively induce or promote an immune response against atleast one dengue virus of at least one serotype in vivo in a subject orex vivo in a population of cells of a subject that is at least aboutequal to or greater than the immune response against the at least onedengue virus of at least one serotype induced or promoted by thecombination of C15/full prM/full E antigenic polypeptides of all fourdengue virus serotypes (e.g., equal to or greater than the combinationof SEQ ID NOS:227-230).

[0260] Some C15/full prM/full E polypeptides of the invention which formVLPs induce an immune response in a subject against at least one denguevirus of at least one serotype that is about equal to or greater thanthat induced against the at least one dengue virus of the at least oneserotype by an incomplete wild-type dengue virus truncated capsid/fullprM/full E VLP (e.g., a VLP formed from wild-type dengue virus C15/fullprM/full E polypeptides), an inactivated dengue virus particle, or both.Select C15/full prM/full E polypeptides also may be able to induce animmune response to at least one dengue virus of at least one serotype adengue virus that is at least equal to or greater than the immuneresponse induced to at least one dengue virus of at least one serotypeby an attenuated WT dengue virus in a subject.

[0261] Especially advantageous are truncated E polypeptides, PRM15/tEpolypeptides, or C15/full prM/full E polypeptides of the invention thatpromote an immune response to at least one dengue virus of all fourknown serotypes that is about equal to or greater than the immuneresponse promoted by one of the corresponding wild-type dengue virustruncated E polypeptide, PRM15/tE polypeptide (e.g., SEQ IDNOS:149-152), or C15/full prM/full E polypeptide (e.g., SEQ IDNOS:227-230) of the same serotype or a combination of such wild-typepolypeptides of all four serotypes. The invention further provides aPRM15/tE polypeptide that promotes a greater immune response to one ormore dengue viruses of all four serotypes than is induced by a wild-typedengue virus PRM15/tE of any serotype (e.g., induces a greater immuneresponse than that induced by a polypeptide consisting essentially ofany of SEQ ID NOS:149-152) or combination thereof. The invention alsoprovides a C15/full prM/full E polypeptide that promotes a greaterimmune response to one or more dengue viruses of all four serotypes thanis induced by a wild-type dengue virus C15/full prM/full E of anyserotype (e.g., induces a greater immune response than that induced by apolypeptide consisting essentially of any of SEQ ID NOS:227-330) orcombination thereof.

[0262] Immune responses generated or induced by the polypeptides of theinvention can be measured by any suitable technique. Examples of usefultechniques in assessing humoral immune responses include flow cytometry,immunoblotting (detecting membrane-bound proteins), including dotblotting, immunohistochemistry (cell or tissue staining), enzymeimmunoassays, immunoprecipitation, immunohistochemistry, RIA(radioimmunoassay), and other EIAs (enzyme immunoassays), such as ELISA(enzyme-linked immunosorbent assay—including sandwich ELISA andcompetitive ELISA) and ELIFA (enzyme-linked immunoflow assay). ELISAassays involve the reaction of a specific first antibody with anantigen. The resulting first antibody-antigen complex is detected byusing a second antibody against the first antibody; the second antibodyis enzyme-labeled and an enzyme-mediated color reaction is produced byreaction with the first antibody. Suitable antibody labels for suchassays include radioisotopes; enzymes, such as horseradish peroxidase(HRP) and alkaline phosphatase (AP); biotin; and fluorescent dyes, suchas fluorescein or rhodamine. Both direct and indirect immunoassays canbe used in this respect. HPLC and capillary electrophoresis (CE) alsocan be utilized in immunoassays to detect complexes of antibodies andtarget substances. General guidance performing such techniques andrelated principles are described in, e.g., Harlow and Lane (1988)ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, NewYork, Hampton R et al. (1990) SEROLOGICAL METHODS A LABORATORY MANUAL,APS Press, St. Paul Minn., Stevens (1995) CLINICAL IMMUNOLOGY ANDSEROLOGY: A LABORATORY PERSPECTIVE, CRC press, Bjerrum (1988) HANDBOOKOF IMMUNOBLOTTING OF PROTEINS, Vol. 2, Zoa (1995) DIAGNOSTICIMMUNOPATHOLOGY: LABORATORY PRACTICE AND CLINICAL APPLICATION, CambridgeUniversity Press, Folds (1998) CLINICAL DIAGNOSTIC IMMUNOLOGY: PROTOCOLSIN QUALITY ASSURANCE AND STANDARDIZATION, Blackwell Science Inc., Bryant(1992) LABORATORY IMMUNOLOGY & SEROLOGY 3rd edition, W B Saunders Co.,and Maddox D E et al. (1983) J. Exp. Med. 158:1211. Specific guidancewith respect to ELISA techniques and related principles are describedin, e.g., Reen (1994) Methods Mol Biol. 32:461-6, Goldberg et al. (1993)Curr Opin Immunol 5(2):278-81, Voller et al. (1982) Lab Res Methods BiolMed 5:59-81, Yolken et al. (1983) Ann N Y Acad Sci 420:381-90, Vaughn etal. (1999) Am J Trop Med Hyg 60(4):693-8, and Kuno et al. J VirolMethods (1991) 33(1-2):101-13. Guidance with respect to Western blottechniques can found in, e.g., Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY (Wiley Interscience Publishers 1995). Specificexemplary applications of Western blot techniques can be found in, e.g.,Churdboonchart et al. (1990) Southeast Asian J Trop Med Public Health21(4):614-20 and Dennis-Sykes et al. (1985) J Biol Stand 13(4):309-14.Specific guidance with respect to flow cytometry techniques is providedin, e.g., Diamond (2000) IN LIVING COLOR: PROTOCOLS IN FLOW CYTOMETRYAND CELL SORTING, Springer Verlag, Jaroszeki (1998) FLOW CYTOMETRYPROTOCOLS, 1st Ed., Shapiro (1995) PRACTICAL FLOW CYTOMETRY, 3rdedition, Rieseberg et al. (2001) Appl Microbiol Biotechnol56(3-4):350-60, Scheffold and Kern (2000) J Clin Immunol 20(6):400-7,and McSharry (1994) Clin Microbiol Rev (4):576-604.

[0263] Briefly, a Western blot assay may be performed by attaching arecombinant dengue antigen, such as a recombinant polypeptide of theinvention, to a nitrocellulose paper and staining with an antibody whichhas a dye attached. Among the methods using a reporter enzyme is the useof a reporter-labeled antihuman antibody. The label may be an enzyme,thus providing an enzyme-linked immunosorbent assay (ELISA). It also maybe a radioactive element, thus providing a radioimmunoassay (RIA).

[0264] Cytotoxic and other T cell immune responses also can be measuredby any suitable technique. Examples of such techniques include ELISpotassay (particularly, IFN-gamma ELISpot), intracellular cytokine staining(ICC) (particularly in combination with FACS analysis), CD8+ T celltetramer staining/FACS, standard and modified T cell proliferationassays, chromium release CTL assay, limiting dilution analysis (LDA),and CTL killing assays. Guidance and principles related to T cellproliferation assays are described in, e.g., Plebanski and Burtles(1994) J Immunol Meth 170:15, Sprent et al. (2000) Philos Trans R SocLond B Biol Sci 355(1395):317-22, and Messele et al. (2000) Clin DiagnLab Immunol 7(4):687-92. LDA is described in, e.g., Sharrock et al.(1990) Immunol Today 11:281-286. ELISpot assays and related principlesare described in, e.g., Czerinsky et al. (1988) J Immunol Meth110:29-36, Olsson et al. (1990) J Clin. Invest 86:981-985, Schmittel etal. (2001) J Immunol Meth 247(1-2):17-24, Ogg and McMichael (1999)Immunol Lett 66(1-3):77-80, Schmittel et al. (2001) J Immunol Meth247(1-2):17-24, Kurane et al. (1989) J Exp Med 170(3):763-75, Chain etal. (1987) J Immunol Meth 99(2):221-8, and Czerkinsky et al. (1988) JImmunol Meth, 110:29-36, as well as U.S. Pat. Nos. 5,750,356 and6,218,132. Tetramer assays are discussed in, e.g., Skinner et al. (2000)J Immunol 165(2):613-7. Other T cell analytical techniques are describedin Hartel et al. (1999) Scand J Immunol 49(6):649-54 and Parish et al.(1983) J Immunol Meth 58(1-2):225-37.

[0265] T cell activation also can be analyzed by measuring CTL activityor expression of activation antigens such as IL-2 receptor, CD69 orHLA-DR molecules. Proliferation of purified T cells can be measured in amixed lymphocyte culture (MLC) assay. MLC assays are known in the art.Briefly, a mixed lymphocyte reaction (MLR) is performed using irradiatedPBMC as stimulator cells and allogeneic PBMC as responders. Stimulatorcells are irradiated (2500 rads) and co-cultured with allogeneic PBMC(1×10⁵ cells/well) in 96-well flat-bottomed microtiter culture plates(VWR) at 1:1 ratio for a total of 5 days. During the last 8 hours of theculture period, the cells were pulsed with 1 uCi/well of ³H-thymidine,and the cells are harvested for counting onto filter paper by a cellharvester as described above. ³H-thymidine incorporation is measured bystandard techniques. Proliferation of T cells in such assays isexpressed as the mean cpm read for the tested wells.

[0266] ELISpot assays measure the number of T-cells secreting a specificcytokine, such as interferon-gamma or tumor necrosis factor-alpha, thatserves as a marker of T-cell effectors. Cytokine-specific ELISA kits arecommercially available (e.g., an IFN-gamma-specific ELISPot is availablethrough R&D Systems, Minneapolis, Minn.). ELISpot assays are furtherdescribed in the Examples section (these and other assays are describedin, e.g., Examples 23-26).

[0267] Where a particular recombinant polypeptide of the invention(e.g., recombinant truncated E polypeptide, full E polypeptide, PRM15/tEpolypeptide, full prM/full E polypeptide, full prM/tE polypeptide, orC15/full prM/full E polypeptide) induces an approximately equal orgreater immune response to a dengue virus of a particular serotype thandoes a corresponding wild-type dengue virus (e.g., truncated Epolypeptide, full E polypeptide, PRM15/tE polypeptide, full prM/full Epolypeptide, full prM/tE polypeptide, or C15/full prM/full Epolypeptide, respectively), such approximately equal or greater immuneresponse is typically an approximately equal or greater humoral immuneresponse. That is, e.g., at least an about equal or higher titer ofneutralizing antibodies to the virus serotype is produced in response tothe recombinant polypeptide than is produced by a correspondingwild-type dengue virus polypeptide (e.g., a wild-type dengue virustruncated E, full E, PRM15/tE, full prM/full E, full prM/tE, or C15/fullprM/full E polypeptide). The particular recombinant truncated E, full E,PRM15/tE polypeptide, full prM/full E, full prM/tE, C15/full prM/full Epolypeptide of the invention desirably induces a T cell response inmammalian cells or a mammal that is substantially similar to (e.g., isat least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100% effective as) the T cell response attendant theadministration or expression of the corresponding wild-type dengue virustruncated E, full E, PRM15/tE, full prM/full E, full prM/tE, or C15/fullprM/full E polypeptide in the mammalian cells or mammal.

[0268] Alternatively, where a T cell response is desired, therecombinant truncated E polypeptide or full E polypeptide, PRM15/tEpolypeptide, or C15/full prM/full E polypeptide can be administered orexpressed with one or more corresponding wild-type dengue virustruncated E polypeptide or full E polypeptide, PRM15/tE polypeptide, orC15/full prM/full E polypeptide dengue prM, E, and/or fragments thereof,respectively (e.g., amino acid sequences comprising known T cellepitopes of such polypeptides), in the mammal. To retain a T cellresponse, the recombinant polypeptide desirably comprises one or morepolypeptide fragments of the capsid protein, prM protein, and E proteinof wild-type DEN-1, DEN-2, DEN-3, and/or DEN-4 of at least about 8 aminoacids in length, and typically about 8 to about 25 amino acids (e.g.,about 10 to about 20 amino acids) in length, wherein such one or morepolypeptide fragments include a T cell epitope of a wild-type denguevirus C protein, prM protein, or E protein. In one preferred aspect, arecombinant PRM15/tE polypeptide comprises T cell epitope sequences, orvariants or mutants of such T cell epitope sequences, that are observedin wild-type dengue virus PRM15 peptides and/or E proteins of two ormore (e.g., multiple) serotypes, and preferably three or four serotypes.In one preferred aspect, a recombinant C15/full prM/full E polypeptidecomprises T cell epitope sequences, or variants or mutants of such Tcell epitope sequences, that are observed in wild-type dengue virus C15peptides, prM proteins, and/or E proteins of two or more (e.g.,multiple) serotypes, and preferably three or four serotypes.

[0269] A preferred characteristic of a recombinant polypeptide of theinvention (including, e.g., a tE or full E polypeptide, PRM15/tEpolypeptide, or C15/full prM/full E polypeptide) is the ability toinduce a neutralizing antibody response against a dengue virus in asubject. A neutralizing antibody response can be determined, e.g., by aplaque reduction neutralization titer (PRNT) assay. Any suitable PRNTassay can be used to determine whether a polypeptide (or polynucleotideexpressing such a polypeptide) induces one or more neutralizingantibodies against one or more dengue viruses of one or more serotypes.An exemplary plaque reduction neutralization titer assay for dengueviruses is described in Russell et al., J Immunol (1967) 99:285-290,which is incorporated herein by reference in its entirety for allpurposes. Other PRNT methods and formats are well known to those ofordinary skill in the art. The results of such an assay depend on theselected level of neutralization desired. A PRNT₅₀, for example, is thehighest serum dilution tested that reduces the number of plaque formingunits (p.f.u.) by at least 50%. Typically, inverse PRNT scores arereported (see the Examples section below for further details onperforming and analyzing the results of such assays). Favorably, asshown herein, select recombinant polypeptides of the invention (andpolynucleotides of the invention expressing such polypeptides) arecapable of inducing a neutralizing antibody response against dengueviruses of at least one or at least two serotypes in a subject.Advantageously, select recombinant polypeptides of the invention (andpolynucleotides of the invention expressing such polypeptides) induce aneutralizing antibody response against one or more dengue viruses ofeach of at least three serotypes. As shown herein, some such recombinantpolypeptides of the invention induce a neutralizing antibody responseagainst one or more dengue viruses of each of the four known wild-typedengue virus serotypes in the subject, such as a mammal.

[0270] In addition to inducing a neutralizing antibody response, arecombinant PRM15/tE polypeptide of the invention also advantageouslyinduces the production of an equal or higher titer of neutralizingantibodies against at least one dengue virus of at least one serotypethan is induced against the at least one dengue virus of the at leastone serotype by at least one wild-type dengue virus PRM15/tE polypeptideof the corresponding serotype (e.g., at least one of SEQ IDNOS:149-152). Such polypeptides also or alternatively induce a highertiter of neutralizing antibodies against at least one dengue virus of atleast one serotype than is induced against at least one dengue virus ofat least one serotype by a combination of wild-type dengue virusPRM15/tE polypeptides of all four known serotypes (e.g., a higher titerof neutralizing antibodies than a combination of four polypeptidesseparately consisting essentially of SEQ ID NOS:149-152). In one aspect,the invention provides a PRM15/tE polypeptide that induces a highertiter of neutralizing antibodies against at least one dengue virus ofeach of the four serotypes in a subject than is induced by a WT denguevirus PRM15/tE polypeptide of any virus serotype (e.g., a higher titerthan a polypeptide consisting essentially of any one of SEQ IDNOS:149-152).

[0271] The invention also provides PRM15/tE polypeptides that arecapable of inducing an equal or higher titer of neutralizing antibodiesagainst at least one dengue virus of each of at least two virusserotypes, preferably at least three virus serotypes, more preferably atleast four virus serotypes, than can be induced antibodies against atleast one dengue virus of each of at least two virus serotypes,preferably at least three virus serotypes, more preferably at least fourvirus serotypes by a WT dengue virus PRM15/tE polypeptide of each of atleast two, at least three, or at least four known serotypes (e.g., atleast two, three, or four of SEQ ID NOS:149-152 or a polypeptideconsisting essentially of one of SEQ ID NOS:149-152).

[0272] In another aspect, in addition to inducing a neutralizingantibody response, a recombinant C15/full prM/full E polypeptide of theinvention also advantageously induces the production of an equal orhigher titer of neutralizing antibodies against at least one denguevirus of at least one serotype than is induced against the at least onedengue virus of the at least one serotype by at least one wild-typedengue virus C15/full prM/full E polypeptide of the correspondingserotype (e.g., at least one of SEQ ID NOS:227-230). Such C15/fullprM/full E polypeptides also or alternatively induce a higher titer ofneutralizing antibodies against at least one dengue virus of at leastone serotype than is induced against at least one dengue virus of atleast one serotype by a combination of wild-type dengue virus C15/fullprM/full E polypeptides of all four known serotypes (e.g., a highertiter of neutralizing antibodies than a combination of four polypeptidesseparately consisting essentially of SEQ ID NOS:227-230). In one aspect,the invention provides a C15/full prM/full E polypeptide that induces ahigher titer of neutralizing antibodies against at least one denguevirus of each of the four serotypes in a subject than is induced by a WTdengue virus C15/full prM/full E polypeptide of any virus serotype(e.g., a higher titer than a polypeptide consisting essentially of anyone of SEQ ID NOS:227-230).

[0273] The invention also provides recombinant C15/full prM/full Epolypeptides that are capable of inducing an equal or higher titer ofneutralizing antibodies against at least one dengue virus of each of atleast two virus serotypes, preferably at least three virus serotypes,more preferably at least four virus serotypes, than can be inducedantibodies against at least one dengue virus of each of at least twovirus serotypes, preferably at least three virus serotypes, morepreferably at least four virus serotypes by a wild-type dengue virusC15/full prM/full E polypeptide of each of at least two, at least three,or at least four known serotypes (e.g., at least two, three, or four ofSEQ ID NOS:227-230 or a polypeptide consisting essentially of one of SEQID NOS:227-230).

[0274] A particularly advantageous feature attendant polypeptides of theinvention is the ability to induce a neutralizing antibody response todengue viruses of all four known virus serotypes in a subject. Anexceptionally beneficial attribute of PRM15tE and C15/full prM/full Epolypeptides of the invention is the ability to induce a higher level ofneutralizing antibodies in a subject against one or more dengue virusesof all four serotypes than the level of neutralizing antibodies inducedagainst such one or more dengue viruses of all four serotypes by acorresponding wild-type dengue virus polypeptide (e.g., wild-typePRM15/tE or wild-type C15/full prM/full E polypeptide) of all fourserotypes (e.g., SEQ ID NOS:19-152 and 227-330, respectively), orcombination of such wild-type polypeptides.

[0275] A particularly desirable characteristic of recombinantpolypeptides of the invention is the ability to induce a neutralizingantibody response to at least one dengue virus, more preferably to atleast two dengue viruses of multiple serotypes, and most preferably todengue viruses of all four virus serotypes, in an animal, including avertebrate, such as, e.g., a mammal. Thus, for example, serum taken froman animal (e.g., mammal) to which an immunogenic or antigenic amount ofa recombinant polypeptide of the invention was administered (or to whichan amount of a recombinant polynucleotide of the invention wasadministered sufficient to express an immunogenic or antigenic amount ofthe recombinant polypeptide) or in which an immunogenic or antigenicamount of a polypeptide of the invention was expressed, diluted at leastabout 30 fold, preferably at least about 40 fold or at least about 50fold, and more preferably at least about 60 fold (e.g., at least about70 fold, 80 fold, or higher), exhibits a neutralizing antibody responseagainst about 50% of the dengue viruses in a sample of dengue viruses ofat least three virus serotypes subjected to a plaque reductionneutralization titer assay.

[0276] An “antigenic amount” is an amount of an antigen, e.g., apolypeptide antigen or polynucleotide encoding such polypeptide antigen,that is sufficient to induce, promote, enhance, or modulate an immuneresponse or immune reaction in cells in vitro, and/or in vivo in asubject or ex vivo in a subject's cells or tissues. An antigenic amountof a polypeptide may be produced by, e.g., administration or delivery ofan antigenic amount of the polypeptide itself, or by administration ordelivery of a polynucleotide that encodes an antigenic amount of suchpolypeptide.

[0277] In another aspect, a recombinant polypeptide of the inventioninduces a neutralizing antibody response to at least one dengue virus ofall four dengue virus serotypes in a mammal to which such recombinantpolypeptide (or polynucleotide encoding such recombinant polypeptide) isadministered without an occurrence of antibody-dependent enhancement(ADE) upon infection of the mammal with the dengue virus. Moreparticularly, the invention provides recombinant polypeptides, whereinserum obtained from an animal (e.g., mammal) to which had beenadministered an antigenic or immunogenic amount of at least one suchrecombinant polypeptide, or at least one recombinant nucleic acid orvector that encodes and/or expresses such an antigenic or immunogenicamount of such recombinant polypeptide of the invention, diluted atleast about 40 fold, about 50 fold, or about 60 fold, and preferably atleast about 70 fold or about 80 fold or more, neutralizes at least about30%, at least about 40%, %, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, or more of a sample comprising atleast one dengue virus of one or more of the four virus serotypes in aplaque reduction neutralization titer assay.

[0278] In one aspect, the invention provides recombinant polypeptidesthat exhibit a reciprocal PRNT₅₀ score of at least about 40, about 50,about 60, or about 70 or higher (e.g., about 40 to about 100, about 40to about 80, about 60 to about 80, or about 70 to about 100) against oneor more dengue viruses of each of two or more and preferably each of allfour dengue virus serotypes. In one preferred aspect, the serum from asubject comprising such a level of polypeptide (either throughexpression or administration), diluted at least about 20fold, 40 fold,60 fold, 70 fold or 80 fold, neutralizes at least about 50% of a sampleof at least one flavivirus in a plaque reduction neutralization titer(PRNT) assay; in one aspect, one of such dilutions neutralizes at leastabout 50% of a sample of a dengue virus of each of the four virusserotypes in a PRNT assay.

[0279] In one aspect of the invention, polypeptides of the inventionthat induce a neutralizing antibody response to at least one denguevirus of all four virus serotypes desirably do so without exhibiting adisproportionate level of neutralizing antibody response to dengueviruses of one serotype such that the immune response or protectionagainst one or more dengue viruses of other serotypes is compromised(i.e., masked by immunodominance). In this respect, reciprocal PRNT₅₀scores against one or more dengue viruses of each of all four serotypesfor sera obtained from a subject comprising an antigenic amount of atleast one recombinant polypeptide of the invention may desirably bewithin a range such that the highest reciprocal PRNT₅₀ score is lessthan about 4×the lowest reciprocal PRNT₅₀ score (e.g., less than about3.9×, about 3.8×, about 3.5×, about 3.4×, or about 3.3× the lowestscore), less than about 3× the lowest reciprocal PRNT₅₀ (e.g., less thanabout 2.9×, about 2.8×, 2.5× about 2.4×, or about 2.3× the lowestscore), and preferably less than about 2× the lowest reciprocal PRNT₅₀score (e.g., less than about 1.9×, about 1.8×, about 1.7×, about 1.6×,about 1.5×, about 1.4×, about 1.3×, about 1.2×, about 1.1× the lowestscore), or from about 4× or less to about 1.5× or less the lowestreciprocal PRNT₅₀ score. Also provided is a composition comprising amixture of two or more recombinant polypeptides of two or moreserotypes, wherein said composition does not show a level ofimmunodominance against one or more dengue viruses of serotypes notincluded in the composition.

[0280] The recombinant polypeptide(s) of the invention can be either asecreted or cell membrane bound (or associated) polypeptide(s). In manyinstances, the recombinant polypeptide desirably is a secretedpolypeptide. For example, in one aspect, some secreted recombinantPRM15/tE polypeptides of the invention surprisingly are secreted moreefficiently than a wild-type dengue virus PRM15/tE polypeptide of atleast one virus serotype (e.g., more efficiently than a polypeptidecomprising or consisting essentially of at least one of SEQ IDNOS:149-152) and more preferably more efficiently than a wild-typePRM15/tE polypeptide of any of the four virus serotypes (e.g., moreefficiently than a polypeptide comprising or consisting essentially ofany of SEQ ID NOS:149-152). Some secreted recombinant C15/full prM/t Epolypeptides of the invention are secreted more efficiently than a WTdengue virus C15/full prM/t E polypeptide of at least one virus serotypeand preferably more efficiently than a WT C15/full prM/t E polypeptideof any of the four virus serotypes.

[0281] Some secreted recombinant C15/full prM/full E polypeptides of theinvention are secreted more efficiently than a WT dengue virus C15/fullprM/full E polypeptide of at least one virus serotype (e.g., moreefficiently than a polypeptide comprising or consisting essentially ofat least one of SEQ ID NOS:227-230) and preferably more efficiently thana WT C15/full prM/full E polypeptide of any of the four virus serotypes(e.g., more efficiently than a polypeptide comprising or consistingessentially of any of SEQ ID NOS:227-230).

[0282] Analysis of polypeptide or protein secretion can be performed byany suitable technique. For example, secretion levels can be determinedby comparing the results of a Western blots/immunoblots performed withthe supernatant of cells transfected with polynucleotides encoding suchpolypeptides and similar supernatants obtained from cells transfectedwith polynucleotides expressing corresponding WT dengue virus, whereboth such recombinant and WT polypeptides are expressed from asubstantially identical expression cassette (e.g., an expressioncassette comprising or consisting essentially of an identical promoter,enhancer, and polyA region sequences). See, e.g., a pMaxVax10.1 vectordescribed below. The use of such a technique to analyze proteinsecretion is provided in the Examples below.

[0283] Measuring the expression level of a recombinant polypeptide ofthe invention (or a corresponding wild-type virus polypeptide forcomparative purposes) can be carried out by any suitable technique.Examples of such techniques include Northern Blot analysis (discussedin, e.g., McMaster et al. (1997) Proc Natl Acad Sci USA 74:4835-38(1977) and Sambrook, infra), reverse transcriptase-polymerase chainreaction (RT-PCR) (as described in, e.g., U.S. Pat. No. 5,601,820 andZaheer et al. (1995) Neurochem Res 20:1457-63, and in situ hybridizationtechniques (as described in, e.g., U.S. Pat. Nos. 5,750,340 and5,506,098). Quantification of proteins also can be accomplished by theLowry assay and other classification protein quantification assays (see,e.g., Bradford (1976) Anal Biochem 72: 248-254 and Lowry et al. (1951) JBiol Chem 193:265). Western blot analysis of recombinant polypeptides ofthe invention obtained from the lysate of cells transfected withpolynucleotides encoding such recombinant polypeptides is a preferredtechnique for assessing levels of recombinant polypeptide expression.The use of such a technique to assess recombinant polypeptide expressionlevels (and wild-type polypeptide expression levels for comparativepurposes) is provided in the Examples below.

[0284] A particularly beneficial characteristic of polypeptides of theinvention, and nucleic acids encoding such polypeptides, is the abilityto induce a protective immune response in a subject, such as an animal,e.g., a mammal (including a primate), against challenge with at leastone dengue virus of at least one serotype. Even more favorably, apolypeptide of the invention induces a protective immune response in asubject against challenge with one or more dengue viruses of each of atleast two virus, at least three, and even at least four virus serotypes.

[0285] The induction of a protective immune response is determined, forexample, by the lack of a disease condition(s) or symptom in a subjectupon or following infection with the at least one dengue virus of the atleast one serotype. In a mouse model, for example, induction of aprotective immune response protects the mice against death usually seenafter injection with a dengue virus. Such mouse models are regularlyused for dengue virus vaccine testing (see, e.g., Johnson and Roehrig,J. Virol. 73(1):783-6 (1999)), although higher primate testing ispreferred (e.g., a rhesus monkey model). In a human, induction of aprotective immune response occurs when there is a detectable lessening,and preferably a complete non-occurrence of DF and/or DHF, uponinfection with a dengue virus (preferably even after repeated infectionwith at least one dengue virus of each of multiple virus serotypes).Typically, though not necessarily, a protective polypeptide of theinvention also induces the production of neutralizing antibodies againstone or more dengue viruses or multiple virus serotypes (e.g., two, threeor four serotypes).

[0286] The polypeptides of the invention can comprise any suitablecombination of the above-described characteristics. For example, in oneaspect, the invention provides a polypeptide (e.g., recombinanttruncated E polypeptide) comprising an amino acid sequence that exhibitsat least about 65%, preferably at least about 75% (e.g., about 80-95%)identity to at least one amino acid sequence selected from the group ofSEQ ID NOS:1-49 and 153-155, wherein the polypeptide induces productionof one or more antibodies that bind to one or more dengue viruses ofeach of the four virus serotypes in a subject more efficiently than anantibody induced by a WT truncated E protein polypeptide of thecorresponding serotype.

[0287] In another aspect, the invention provides a polypeptide (e.g.,recombinant or synthetic PRM15/tE polypeptide) comprising an amino acidsequence that exhibits at least about 65%, at least about 75%,preferably at least about 85%, 90%, or 95% amino acid sequence identityto at least one sequence selected from any of SEQ ID NOS:65-116, whereinthe polypeptide induces production of one or more antibodies that bindto one or more dengue viruses of each of the four serotype moreefficiently than at least one antibodies induced by any WT dengue viruspolypeptides selected from SEQ ID NOS:149-152.

[0288] In another aspect, the invention provides a polypeptide (e.g.,recombinant C15/full prM/full E polypeptide) comprising an amino acidsequence that exhibits at least about 65%, at least about 75% (e.g.,about 80-95%), and/or preferably at least about 85% or 90% identity toat least one amino acid sequence selected from the group of SEQ IDNOS:39-148, and 236-253 (or selected from the group of SEQ IDNOS:39-145, 147-148 and 236-253 or any other group comprising acombination of two or more of these polypeptides), wherein thepolypeptide induces production of antibodies that bind to one or moredengue viruses of each of the four virus serotypes in a subject moreefficiently than at least one antibodies induced by any WT dengue viruspolypeptide selected from SEQ ID NOS:227-230.

[0289] Recombinant tE polypeptides are desirably associated or extendedwith an ER-targeting signal amino acid sequence, which typically hassubstantially sequence identity with a C-terminal flaviviral prM aminoacid sequence, and preferably has substantially identity with (or isselected from) (e.g., having at least about 75%, 80%, 85%, 86%, 87%, 88%or 89%, preferably at least about 90%, 91%, 92%, 93%, or 94%, and morepreferably at least about 95% (e.g., about 87-95%), 96% 97%, 98%, 99%,99.5% sequence identity) a dengue virus PRM15 sequence or a novelhomolog thereof (e.g., a sequence selected from any of SEQ IDNOS:52-64), as described above, thereby forming a signal peptide/tEpolypeptide, such as, e.g., a PRM15/tE polypeptide (e.g., any of SEQ IDNOS:1-49 and 153-155). Such a recombinant tE polypeptide or PRM15/tEpolypeptide can be extended with a C terminal E protein fragmentpolypeptide as described above, such as, e.g., a sequence substantiallyidentical (e.g., having at least about 75%, 80%, 85%, 86%, 87%, 88% or89%, preferably at least about 90%, 91%, 92%, 93%, or 94%, and morepreferably at least about 95% (e.g., about 87-95%), 96% 97%, 98%, 99%,99.5% sequence identity) with any of SEQ ID NOS:127-136 to form arecombinant full E polypeptide or PRM15/full E polypeptide. In additionor alternatively, a recombinant PRM15/tE polypeptide or a PRM15/full Epolypeptide of the invention can be extended with an N terminalC15/truncated prM polypeptide as described above, such as, e.g., asequence substantially identical (e.g., having at least about 75%, 80%,85%, 86%, 87%, 88% or 89%, preferably at least about 90%, 91%, 92%, 93%,or 94%, and more preferably at least about 95% (e.g., about 87-95%), 96%97%, 98%, 99%, 99.5% sequence identity) with any one of SEQ IDNOS:117-126, to form a recombinant C15/full prM/tE or C15/full prM/fullE polypeptide, respectively. Some such recombinant polypeptides areexpressed and/or secreted at higher levels than prM/E fusion proteinsformed from WT DEN sequences, as shown above and in Examples below.

[0290] Some such recombinant tE, full E, PRM15/tE, PRM15/full E,C15/full prM/full E, and C15/full prM/tE polypeptides also desirablyexhibit the ability to induce a neutralizing antibody response againstat least one dengue virus of at least one serotype, and preferablyagainst one or more dengue viruses of multiple serotypes, in a subject,e.g., mammalian host. As noted above, some such polypeptides induce ahigher neutralizing antibody titer against at least one dengue virus ofat least one serotype than is induced against such at least one denguevirus of at least one serotype by a wild-type dengue polypeptide ofsimilar size and configuration of the same or similar serotype (e.g.,wild-type dengue virus tE, full E, PRM15/tE, PRM15/full E, C15/fullprM/full E, C15/full prM/tE), as described above. Some such recombinanttE, full E, PRM15/tE, PRM15/full E, C15/full prM/full E, and C15/fullprM/tE polypeptides also are able to induce a protective immune responseagainst challenge by at least one dengue virus of at least one serotypein a subject host, and preferably exhibit the ability to induce aprotective immune response against challenge by at one two dengue virusof each of at least two serotypes, in a subject host. Some suchrecombinant tE, full E, PRM15/tE, PRM15/full E, C15/full prM/full E, andC15/full prM/tE polypeptides of the invention are able to induce aprotective immune response against challenge by at least one denguevirus of each DEN-1, DEN-2, DEN-3, and DEN-4 in a subject host.Desirably, such polypeptides induce such neutralizing antibody responsesand/or protective immune responses against one or more viruses of two ormore dengue virus serotypes without the occurrence of ADE.

[0291] In another preferred aspect, the invention provides a recombinanttE polypeptide comprising an amino acid sequence that has at least about65% amino acid sequence identity, at least about 75% (e.g., about80-95%), preferably at least about 85%, or at least about 90% or atleast about 95% amino acid sequence identity with at least one aminoacid sequence selected from the group of SEQ ID NOS:1-49 and 153-155,wherein the polypeptide induces a neutralizing antibody response to oneor more dengue viruses of at least two virus serotypes in a subject.Preferably, such polypeptides induce a neutralizing antibody response toone or more dengue viruses of each of at least three virus serotypes,and, even more preferably, against one or more dengue viruses of each ofdengue-1, dengue-2, dengue-3, and dengue-4 serotypes in the subject.Optionally, such neutralizing antibody (Ab) production produces higherneutralizing Ab titers than are obtained with a corresponding wild-typetruncated E of one or more virus serotypes. These polypeptides also canbe associated with any of the above-described characteristics, orcombinations thereof, attendant polypeptides of the invention (e.g.,inclusion of an ER-targeting sequence, inclusion of at least one Cterminal E protein fragment polypeptide and/or at least one N terminalC15/truncated prM polypeptide, higher secretion, higher expression, andinduction of a protective immune response in a subject such as a mammalto one or more dengue viruses of multiple serotypes without induction ofADE). Such polypeptides exhibit one or more of the characteristics ofthe polypeptides of the invention (e.g., ability to induce an immuneresponse against at least one dengue virus of one or more serotypes;ability to induce an immune response against at least one dengue virusof one or more serotypes that is greater than that induced by acorresponding WT dengue polypeptide; ability to induce the production ofneutralizing antibodies to one or more dengue viruses of multipleserotypes in a subject) or any suitable combination thereof. Suchpolypeptides are useful in methods of the invention described herein,including methods of inducing an immune response against at least onedengue virus of at least one serotype, methods of inducing a protectiveimmune response against a dengue virus, and/or methods of detecting thepresence of antibodies against dengue viruses of one or more serotypesin a sample.

[0292] In another aspect of the invention, the polypeptide comprises anamino acid sequence that has substantially identity (e.g., having atleast about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity) withat least one of SEQ ID NOS:65-116. Preferred polypeptides comprise asequence selected from any of SEQ ID NOS:65-116. Such polypeptides cancomprise any of the above-described characteristics attendantpolypeptides of the invention (e.g., ability to induce an immuneresponse against at least one dengue virus of one or more serotypes;ability to induce an immune response against at least one dengue virusof one or more serotypes that is greater than that induced by acorresponding WT dengue polypeptide; ability to induce the production ofneutralizing antibodies to one or more dengue viruses of multipleserotypes in a subject) or any suitable combination thereof, and areuseful in methods of inducing an immune response against at least onedengue virus of at least one serotype, methods of inducing a protectiveimmune response against a dengue virus, and/or methods of detecting ordiagnosing the presence of antibodies against dengue viruses of one ormore serotypes in a sample.

[0293] In yet another aspect, the invention provides a polypeptidecomprising an amino acid sequence that has substantial sequence identity(e.g., at least about 65% amino acid sequence identity, desirably atleast about 75% amino acid sequence identity, favorably at least about80% or at least about 85% amino acid sequence identity, and preferablyat least about 90% or at least about 95% amino acid sequence identity)with a sequence selected from any of SEQ ID NOS:139-148, 236-253, 343,and 345, or selected from any of SEQ ID NOS:139-145, 147-148, 236-253,343, and 345. Preferred polypeptides in this aspect comprise a sequenceselected from the group of SEQ ID NOS:139-148, 236-253, 343, and 345.Such polypeptides can comprise any of the above-describedcharacteristics attendant polypeptides of the invention (e.g., abilityto induce an immune response against at least one dengue virus of one ormore serotypes; ability to induce an immune response against at leastone dengue virus of one or more serotypes that is greater than thatinduced by a corresponding WT dengue polypeptide; ability to induce theproduction of neutralizing antibodies to one or more dengue viruses ofmultiple serotypes in a subject) or any suitable combination thereof,and are useful in methods of inducing an immune response against atleast one dengue virus of at least one serotype, methods of inducing aprotective immune response against a dengue virus, and/or methods ofdetecting or diagnosing the presence of antibodies against dengueviruses of one or more serotypes in a sample.

[0294] In another aspect, the invention provides a recombinant truncatedE polypeptide encoded by a nucleic acid comprising a polynucleotidesequence selected from the group of: (a) a polynucleotide sequencehaving at least about 85% sequence identity to at least onepolynucleotide sequence selected from the group of SEQ ID NOS:285-330 ora complementary polynucleotide sequence thereof; (b) a RNApolynucleotide sequence comprising a DNA sequence selected from thegroup of SEQ ID NOS:285-330 in which all of the thymine nucleotideresidues in the DNA sequence are replaced with uracil nucleotideresidues or a complementary RNA polynucleotide sequence thereof; (c) aRNA polynucleotide sequence that has at least about 85% sequenceidentity to at least one RNA polynucleotide sequence of (b) or acomplementary RNA polynucleotide sequence thereof; (d) a polynucleotidesequence that hybridizes under at least stringent conditions oversubstantially the entire length of a polynucleotide sequence of (a)-(c);(e) a polynucleotide sequence which would hybridize under at leaststringent conditions over substantially the entire length of apolynucleotide sequence of any of (a)-(d) but for the degeneracy of thegenetic code; and (f) a polynucleotide sequence that possesses anycombination of the features of the polynucleotide sequences of (a)-(e).

[0295] In yet another aspect, the invention provides a recombinantPRM15/tE polypeptide encoded by a nucleic acid comprising apolynucleotide sequence selected from the group of: (a) a polynucleotidesequence having at least about 85% sequence identity to at least onepolynucleotide sequence selected from the group of SEQ ID NOS:156-200and 235, or a complementary polynucleotide sequence thereof; (b) a RNApolynucleotide sequence comprising a DNA sequence selected from thegroup of SEQ ID NOS:156-200 and 235 in which all of the thyminenucleotide residues in the DNA sequence are replaced with uracilnucleotide residues or a complementary RNA polynucleotide sequencethereof; (c) a RNA polynucleotide sequence that has at least about 85%,90%, or 95% sequence identity to at least one RNA polynucleotidesequence of (b) or a complementary RNA polynucleotide sequence thereof;(d) a polynucleotide sequence that hybridizes under at least stringentconditions over substantially the entire length of a polynucleotidesequence of (a)-(c); (e) a polynucleotide sequence which would hybridizeunder at least stringent conditions over substantially the entire lengthof a polynucleotide sequence of any of (a)-(d) but for the degeneracy ofthe genetic code; and (f) a polynucleotide sequence that possesses anycombination of the features of the polynucleotide sequences of (a)-(e).Such polypeptides exhibit any of the above-described characteristicsattendant polypeptides of the invention and are useful in methods of theinvention, e.g., methods of inducing an immune response against at leastone dengue virus of at least one serotype, and/or methods of detectingor diagnosing the presence of antibodies against dengue viruses of oneor more serotypes in a sample.

[0296] In another aspect, the invention includes a recombinant C15/fullprM/full E polypeptide encoded by a nucleic acid comprising apolynucleotide sequence selected from the group of: (a) a polynucleotidesequence having at least about 85%, 90%, or 95% sequence identity to atleast one polynucleotide sequence selected from the group of SEQ IDNOS:201-210, 254-271, 342, and 344, or a complementary polynucleotidesequence thereof; (b) a RNA polynucleotide sequence comprising a DNAsequence selected from the group of SEQ ID NOS:201-210, 254-271, 342,and 344,in which all of the thymine nucleotide residues in the DNAsequence are replaced with uracil nucleotide residues or a complementaryRNA polynucleotide sequence thereof; (c) a RNA polynucleotide sequencethat has at least about 85% sequence identity to at least one RNApolynucleotide sequence of (b) or a complementary RNA polynucleotidesequence thereof; (d) a polynucleotide sequence that hybridizes under atleast stringent conditions over substantially the entire length of apolynucleotide sequence of (a)-(c); (e) a polynucleotide sequence whichwould hybridize under at least stringent conditions over substantiallythe entire length of a polynucleotide sequence of any of (a)-(d) but forthe degeneracy of the genetic code; and (f) a polynucleotide sequencethat possesses any combination of the features of the polynucleotidesequences of (a)-(e). Such polypeptides exhibit any of theabove-described characteristics attendant polypeptides of the inventionand are useful in methods of the invention, e.g., methods of inducing animmune response against at least one dengue virus of at least oneserotype, and/or methods of detecting or diagnosing the presence ofantibodies against dengue viruses of one or more serotypes in a sample.

[0297] Recombinant polypeptides of the invention advantageously arecapable of inducing an immune response to one or more dengue viruses ofat least one, preferably at least two, more preferably at least three,and most preferably at least all four virus serotypes in a subject oversustained periods of time. For example, delivery or administration of anantigenic or immunogenic amount of at least one polypeptide of theinvention to a subject induces a neutralizing antibody immune responseto at least one dengue virus of at least one serotype for a period of atleast about 30 days, at least about 40 days, desirably at least about50, favorably at least about 70 or about 80 days, preferably at leastabout 100 days, more preferably at least about 120 days, and even morepreferably at least about 180 days (e.g., about 3, 4, 6, or 9 months,about 1 year, 2 years, or longer) following delivery or administrationof the at least one polypeptide to the subject.

[0298] In another aspect, delivery or administration of at least onepolypeptide of the invention by delivery or administration of a suitablenucleic acid vector (e.g., a pMaxVax10.1 vector) comprising at least onepolynucleotide encoding an antigenic or immunogenic amount of the atleast one polypeptide, induces a neutralizing antibody immune responseto at least one dengue virus of at least one serotype for a period of atleast about 30 days, at least about 40 days, desirably at least about50, favorably at least about 70 or about 80 days, preferably at leastabout 100 days, more preferably at least about 120 days, and even morepreferably at least about 180 days (e.g., about 9 months, about 1 year,2 years, or longer) after initial expression of the at least onepolypeptide in a subject.

[0299] An immune response obtained by administration of a recombinantpolypeptide of the invention (or polynucleotide or vector coding onexpression for such a polypeptide, examples of which are furtherdiscussed herein), such as, e.g., a recombinant chimeric dengue virusPRM15/tE or C15/full prM/full E polypeptide, desirably lasts longer thanthe immune response induced by administration of a correspondingwild-type dengue virus polypeptide (e.g., wild-type dengue virusPRM15/tE or C15/full prM/full E polypeptide) or polynucleotide or vectorcoding on expression of such a wild-type dengue virus polypeptide.

[0300] In another aspect, the invention provides a recombinant orchimeric polypeptide including at least about 3, least about 5, at leastabout 6, at least about 7, at least about 8, at least about 10, at leastabout 15, at least about 20, at least about 25, at least about 30 ormore amino acid (residue) sequence fragments of at least about 8 aminoacid residues in length, at least about 10 amino acid residues inlength, or at least about 15 amino acids in length, wherein such aminoacid fragments are observed in one or more wild-type DEN-1, DEN-2,DEN-3, and DEN-4 prM/E amino acid sequences, and wherein suchpolypeptide comprises at least about 2 (preferably at least about 3, atleast about 4, at least about 5, at least about 10, at least about 15,or more) non-contiguous fragments from any one wild-type dengue virusprM/E sequence. Such recombinant or chimeric polypeptides also oralternatively can promote or enhance an immune response, especially inthe case of a humoral immune response, about equal to or greater than arecombinant or chimeric dengue virus antigen and/or flavivirus antigenconsisting of 4 or less distinct dengue virus and/or flavivirus aminoacid sequence fragments.

[0301] In one aspect, for example, the invention provides a recombinantor chimeric polypeptide comprising at least about 5 to about 20 aminoacid sequence fragments comprising at least about 10 to about 40 aminoacid residues in length, wherein such amino acid sequence fragments arenon-contiguous fragments of one or more wild-type DEN-1, DEN-2, DEN-3,and DEN-4 prM/E amino acid sequences.

[0302] Nucleic Acids

[0303] The invention further provides a nucleic acid comprising anucleotide sequence encoding any of the above-described polypeptides ofthe invention, including recombinant and chimeric tE, full length E,PRM15/tE, PRM15/full E, C15/full prM/full E, and C15/full prM/tEpolypeptides of the invention. The terms “nucleic acid” and“polynucleotide” are synonymously used throughout in reference to suchrecombinant dengue antigen-encoding DNA, RNA, or other novel nucleicacid molecules of the invention, unless otherwise stated or clearlycontradicted by context. The nucleic acid encoding a recombinant,synthetic, mutant, and/or isolated polypeptide of the invention can beany type of nucleic acid suitable for expressing a polypeptide of theinvention (e.g., single stranded or double stranded RNA, DNA, orcombinations thereof) and can include any suitable nucleotide base, baseanalog, and/or backbone (e.g., a backbone formed by, or including, aphosphothioate, rather than phosphodiester, linkage). Modifications tothe nucleic acid are particularly tolerable in the 3rd position of anmRNA codon sequence encoding such a polypeptide. Examples of modifiednucleotides that can be incorporated in the polynucleotide sequence areprovided in, e.g., the MANUAL OF PATENT EXAMINING PROCEDURE §2422 (7thRevision—2000). Additional and alternative sequence modifications aredescribed elsewhere herein. In some aspects, a nucleic acid of theinvention may be an isolated nucleic acid. A nucleic acid of theinvention may be termed a recombinant, synthetic, and/or mutant nucleicacid or polynucleotide; a recombinant, synthetic, and/or mutant nucleicacid or polynucleotide of the invention is often simply referred to as a“recombinant nucleic acid,” “recombinant polynucleotide,” or even moresimply a “nucleic acid” or “polynucleotide.” A nucleic acid of theinvention may be referred to as a recombinant or synthetic dengueantigen-encoding nucleic acid.

[0304] In another aspect, a recombinant nucleic acid of the inventioncomprises any nucleotide sequence that results in the production (e.g.,suitable production) of a recombinant polypeptide of the invention uponexpression or translation in a desired host cell. Suitable production ofa recombinant polypeptide of the invention may be the expression of animmunogenic amount or antigenic amount of the polypeptide of theinvention. An “immunogen” is a molecule that induces, promotes,enhances, or modulates an immune response (e.g., in an in vitrocell-based assay or in vivo in a subject to which the immunogen isadministered or ex vivo in cells (e.g., a subject's cells or tissue)that are transplanted into the subject). In one particular aspect, an“immunogen” is an antigen that elicits an immune response. In certainparticular aspects, an immunogen is an antigen that elicits a strongimmune response, particularly in the context of protective immunity toat least one pathogen (e.g., at least one flavivirus of at least oneserotype). An “immunogenic amount” is typically an amount of a molecule,e.g., a polypeptide or polynucleotide, that is sufficient to induce,promote, enhance, or modulate an immune response in cells in vitro,and/or in vivo in a subject or ex vivo in a subject's cells ortissue(s). In one aspect, an immunogenic amount refers to an amount ofan antigen that elicits an immune response that provides at leastpartial or complete protective immunity against at least one pathogen,such as a flavivirus (e.g., such as against at least one dengue virus ofone or more serotypes). An immunogenic amount of a polypeptide may beproduced by, e.g., administration or delivery of an immunogenic amountof the polypeptide itself to cells in vitro or to a population of cellsor tissue(s) of a subject ex vivo or in vivo, or by administration ordelivery of a nucleic acid that encodes an immunogenic amount of suchpolypeptide to cells in vitro or to a population of cells or tissue(s)of a subject ex vivo or in vivo.

[0305] Nucleic acids of the invention encode polypeptides of theinvention that have one or more of the properties described above.Nucleic acids of the invention are similarly useful in methods of theinvention, including a method of inducing an immune response against atleast one flavivirus of at least one serotype (e.g., preferably, denguevirus) in a subject that comprises contacting, administering, ordelivering a nucleic acid encoding at least one polypeptide of theinvention to such subject (or to population of cells of the subject) andmethods of inducing a protective immune response against at least oneflavivirus of one or more serotypes (e.g., preferably, at least onedengue virus of two or more serotypes) in a subject that comprisescontacting, administering, or delivering a nucleic acid encoding atleast one polypeptide of the invention to such subject (or to apopulation of cells thereof).

[0306] As such, a nucleic acid of the invention is not limited to anucleotide sequence that directly codes for expression or production ofa polypeptide of the invention. For example, the nucleic acid cancomprise a nucleotide sequence which results in a polypeptide of theinvention through intein-like expression (as described in, e.g., Colsonand Davis (1994) Mol Microbiol 12(3): 959-63, Duan et al. (1997) Cell89(4):555-64, Perler (1998) Cell 92(1):1-4, Evans et al (1999)Biopolymers 51(5):333-42, and de Grey, Trends Biotechnol (2000)18(9):394-99), or a nucleotide sequence which comprises self-splicingintrons (or other self-spliced RNA transcripts), which form anintermediate recombinant polypeptide-encoding sequence (as described in,e.g., U.S. Pat. No. 6,010,884). The polynucleotides also can comprisesequences which result in other splice modifications at the RNA level toproduce an mRNA transcript encoding the polypeptide and/or at the DNAlevel by way of trans-splicing mechanisms prior to transcription(principles related to such mechanisms are described in, e.g., Chabot,Trends Genet (1996) 12(11):472-78, Cooper (1997) Am J Hum Genet61(2):259-66, and Hertel et al. (1997) Curr Opin Cell Biol 9(3):350-57).Due to the inherent degeneracy of the genetic code, several nucleicacids can code for any particularly polypeptide of the invention. Thus,for example, any of the particular recombinant dengue-antigen-encodingnucleic acids described herein can be modified by replacement of one ormore codons with an equivalent codon (with respect to the amino acidcalled for by the codon) based on genetic code degeneracy.

[0307] Polynucleotides of the invention can be obtained by applicationof any suitable synthesis, manipulation, and/or isolation techniques, orcombinations thereof. For example, polynucleotides of the invention aretypically and preferably produced through standard nucleic acidsynthesis techniques, such as solid-phase synthesis techniques known inthe art. In such techniques, fragments of up to about 100 bases usuallyare individually synthesized, then joined (e.g., by enzymatic orchemical ligation methods, or polymerase mediated recombination methods)to form essentially any desired continuous nucleic acid sequence. Thesynthesis of the nucleic acids of the invention can be also facilitated(or alternatively accomplished), by chemical synthesis using, e.g., theclassical phosphoramidite method, which is described in, e.g., Beaucageet al. (1981) Tetrahedron Letters 22:1859-69, or the method described byMatthes et al. (1984) EMBO J 3:801-05, e.g., as is typically practicedin automated synthetic methods. The polynucleotide of the inventiondesirably by the phosphoramidite technique also preferably isaccomplished by way of an automatic DNA synthesizer. Other techniquesfor synthesizing nucleic acids and related principles are described in,e.g., Itakura et al. (1984) Annu Rev Biochem 53:323, Itakura et al.(1984) Science 198:1056, and Ike et al. (1983) Nucl Acid Res 11:477.

[0308] Conveniently, custom made nucleic acids can be ordered from avariety of commercial sources, such as The Midland Certified ReagentCompany (mcrc@oligos.com), the Great American Gene Company(http://www.genco.com), ExpressGen Inc. (www.expressgen.com), OperonTechnologies Inc. (Alameda, Calif.). Similarly, custom peptides andantibodies can be custom ordered from any of a variety of sources, e.g.,PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc.(http://www.htibio.com), and BMA Biomedicals Ltd. (U.K.), Bio.Synthesis,Inc.

[0309] Recombinant DNA techniques useful in modification of nucleicacids are well known in the art (e.g., restriction endonucleasedigestion, ligation, reverse transcription and cDNA production, andPCR). Useful recombinant DNA technology techniques and principlesrelated thereto are provided in, e.g., Mulligan (1993) Science260:926-932, Friedman (1991) THERAPY FOR GENETIC DISEASES, OxfordUniversity Press, Ibanez et al. (1991) EMBO J 10:2105-10, Ibanez et al.(1992) Cell 69: 329-41 (1992), and U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648, and are more particularly described inSambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, ColdSpring Harbor Press, and the third edition thereof (2001), Ausubel etal. (1994-1999), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, WileyInterscience Publishers (with Greene Publishing Associates for someeditions), Berger and Kimmel, “Guide to Molecular Cloning Techniques” inMeth Enzymol, 152, Acad. Press, Inc. (San Diego, Calif.), and Watson etal., RECOMBINANT DNA (2d ed.).

[0310] As described in more detail herein, recombinant nucleic acids ofthe invention include: (1) polynucleotide sequences that encode any ofthe above-described polypeptide sequences and immunogenic or antigenicfragments thereof (including, e.g., recombinant immunogenic tE, full E,PRM15/tE, PRM15/full E, C15/full prM/full E, and C15/full prM/tEpolypeptides of the invention), and complementary sequences thereof; (2)polynucleotide sequences complementary to such polynucleotide sequencesof (1) (and fragments thereof); polynucleotides that hybridize under atleast stringent conditions to the nucleic acid sequences defined herein,including any polynucleotide sequence of (1) and (2), and complementarysequences thereof; (3) novel immunogenic or antigenic fragments of allsuch nucleic acid sequences, including those described in (1) and (2)above, which have one or more of the immune-response inducing propertiesset forth herein or encode one or more polypeptides have one or more ofthe immune-response inducing properties set forth herein, andcomplementary sequences of such fragments; and (4) variants, analogs,and homologue derivatives of all of the above.

[0311] The polynucleotides of the invention can be double-stranded orsingle-stranded, and if single-stranded, can be the coding strand or thenon-coding (i.e., antisense or complementary) strand. In addition to anucleotide sequence encoding a polypeptide of the invention, thepolynucleotide of the invention can comprise one or more additionalcoding nucleotide sequences, so as to encode, e.g., a fusion protein, apre-protein, a prepro-protein, a heterologous transmembrane domain,targeting sequence (other than a signal sequence), or the like (moreparticular examples of which are discussed further herein), and/or cancomprise non-coding nucleotide sequences, such as introns, or 5′ and/or3′ untranslated regions effective for expression of the coding sequencein a suitable host.

[0312] For example, a polynucleotide of the invention that encodes atruncated E or full E protein (i.e., tE-encoding or full E-encodingpolynucleotide) may further comprise a nucleotide sequence that encodesan ER-targeting signal sequence positioned near to, or fused to, thetE-encoding or full E-encoding immunogenic sequence of the invention.Preferably, the signal sequence-encoding nucleic acid sequence encodes asignal sequence having the preferred features of signal sequencesdescribed above (e.g., a 15-amino acid fragment of the C terminal of aprM protein of a flavivirus, preferably a dengue virus, or a sequencehaving substantial identity therewith).

[0313] In one aspect, the invention provides a nucleic acid comprising asignal sequence-encoding sequence having substantial nucleotide sequenceidentity (e.g., at least about 65%, 70%, 75%, preferably at least about80% or 85%, and more preferably at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity) with at least one of SEQ IDNOS:272-284, and preferably SEQ ID NOS:272-280. Such polynucleotidesequences, which encode signal peptides, are similarly useful as and inapplications including the signal peptides of the invention discussedabove. In one aspect, truncated E polypeptide-encoding or full length Epolypeptide-encoding nucleic acids comprising a sequence selected fromthe group of SEQ ID NOS:272-284 are also a feature of the presentinvention.

[0314] The invention also provides novel nucleic acids useful in theproduction of recombinant dengue virus antigens and other applications(e.g., for use in methods of inducing an immune response against one ormore dengue viruses and/or in therapeutic or prophylactic methods, asvaccines, in diagnostic methods and systems, as nucleic acid probes, inthe amplification of smaller nucleic acid sequences that encodeimmunogenic fragments of such recombinant dengue virus antigens (suchuses are discussed elsewhere herein)).

[0315] For example, in one respect, the invention provides a nucleicacid comprising a polynucleotide sequence that has substantial sequenceidentity (e.g., at least about 65%, 70%, 75%, preferably at least about80% or 85%, and more preferably at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity) with the polypeptide sequence of atleast one of SEQ ID NOS:285-330. In particular aspects, the nucleic acidcomprises a sequence selected from the group of SEQ ID NOS:285-330. Sucha nucleic acid encodes at least a recombinant truncated E polypeptide ofthe invention and is generally termed a recombinant tEpolypeptide-encoding nucleic acid. Such nucleic acids having one or moreof the properties of recombinant nucleic acids described below.

[0316] Nucleic acids consisting of and/or consisting essentially of suchsequences such as the group of SEQ ID NOS:285-330 encode a polypeptideof a length approximately equal to a truncated E sized recombinantdengue antigen of the invention, as discussed above. Such nucleic acidsare typically at least about 1300 nucleotides in length, and typicallyare about 1300-1375 nucleotides in length (e.g., about 1340 nucleotidesin length).

[0317] The invention also provides a nucleic acid comprising firstnucleotide sequence encoding recombinant truncated E polypeptide dengueantigens and a second nucleotide sequence encoding a signal peptide. Forexample, in one aspect, the invention provides a nucleic acid comprisinga sequence that has substantial identity (e.g., at least about 75%, 80%,85%, 86%, 87%, 88% or 89%, preferably at least about 90%, 91%, 92%, 93%,or 94%, and more preferably at least about 95% (e.g., about 87-95%), 96%97%, 98%, 99%, 99.5% sequence identity) with at least one of SEQ IDNOS:156-200 and 235. More desirably, the polynucleotide comprises asequence selected from the group of SEQ ID NOS:156-200 and 235. Suchnucleic acids are typically at least about 1350 nucleotides in length,and more typically are about 1350-1400 nucleotides in length (e.g.,about 1385 nucleotides in length). Such a nucleic acid encodes a PRM15/tE polypeptide and is generally termed a PRM15/tE polypeptide-encodingnucleic acid. In particular aspects, the nucleic acid comprises asequence selected from the group of SEQ ID NOS:157-159, 185, 187, 172,200, and 235. Such nucleic acids having one or more of the properties ofrecombinant nucleic acids are described below.

[0318] The invention also provides a nucleic acid sequence that hassubstantial sequence identity (e.g., at least about 65%, 70%, 75%,preferably at least about 80% or 85%, and more preferably at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with thepolypeptide sequence of at least one of SEQ ID NOS:211-214. Inparticular aspects, the nucleic acid comprises a sequence selected fromthe group of SEQ ID NOS:211-214. These nucleotide sequences are humancodon optimized nucleotide sequences encoding DEN-1, DEN-2, DEN-3, andDEN-4 PRM15/tE polypeptides, respectively, and are generally termedhuman CO WT PRM15/tE polypeptide-encoding nucleic acids. Thepolypeptides encoded by these nucleic acid sequences offer improvedbiological properties over non human codon-optimized (CO) wild-typePRM15/tE polypeptide-encoding nucleotide sequences. Such polynucleotidesare markedly different in structure and lack substantial identity withnon human CO wild-type PRM15/tE polypeptide-encoding nucleotidesequences. For example, the polypeptide encoded by these sequences areexpressed at higher levels and/or are secreted at higher levels thansimilar PRM15/tE polypeptides expressed from non human CO WT denguevirus PRM15/tE sequences.

[0319] Also included is a nucleic acid sequence that has substantialsequence identity (e.g., at least about 75%, 80%, 85%, 86%, 87%, 88% or89%, preferably at least about 90%, 91%, 92%, 93%, or 94%, and morepreferably at least about 95% (e.g., about 87-95%), 96% 97%, 98%, 99%,99.5% sequence identity) with the polypeptide sequence of at least oneof SEQ ID NOS:215-218. Some such nucleic acid comprises a sequence of atleast about 1800 nucleotides. In particular aspects, the nucleic acidcomprises a sequence selected from the group of SEQ ID NOS:215-218.These nucleic acid sequences are human codon optimized nucleotidesequences that encode DEN-1, DEN-2, DEN-3, and DEN-4 C15/full prM/full Epolypeptides, respectively, and are termed human CO WT C15/full prM/fullE polypeptide-encoding nucleic acids. These encoded polypeptides exhibitimproved biological properties compared to C15/full prM/full Epolypeptides expressed from non human CO wild-type C15/full prM/full Epolypeptide-encoding nucleic acids. For example, the polypeptidesencoded by these sequences are expressed at higher levels and/or aresecreted at higher levels than similar C15/full prM/full E polypeptidesexpressed from non human CO wild-type C15/full prM/full Epolypeptide-encoding nucleic acid sequences.

[0320] The invention also provides a recombinant nucleic acids thatencode, e.g., antigenic fusion proteins each comprising: (1) a C15dengue virus signal sequence (which also includes a Met residue as thefirst residue of the signal sequence, thereby forming a 16-amino acidsignal sequence); (2) a full length prM dengue virus sequence; and 3) afull length envelope (E) protein sequence, wherein these three sequencesare fused together in the order 1, 2, and 3. In another aspect, theinvention provides recombinant nucleic acids encoding antigenic fusionproteins that each comprise a full length prM dengue virus sequencefused to a full length envelope (E) protein sequence.

[0321] In another aspect, the invention provides a nucleic acidcomprising at least a first polynucleotide sequence comprising a tEpolypeptide-encoding polynucleotide sequence or a PRM15/tEpolypeptide-encoding polynucleotide sequence and at least a secondpolynucleotide sequence that encodes a polypeptide sequence that has atleast about 55%, preferably at least about 65%, and more preferably atleast about 75% (e.g., at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, or more) amino acid sequence identitywith a sequence selected from the group of SEQ ID NOS:127-136.Alternatively, the second polynucleotide sequence that has at leastabout 55%, preferably at least about 65%, and more preferably at leastabout 75% (e.g., at least about 80%, at least about 85%, at least about90%, at least about 95%, or more) nucleotide identity with a sequenceselected from the group of SEQ ID NOS:223-226. In some aspects, it isdesirable that such nucleic acids comprise a sequence selected from thegroup of SEQ ID NOS:223-226.

[0322] In another aspect, the invention provides a nucleic acidcomprising a polynucleotide sequence that has substantial sequenceidentity (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with the polypeptidesequence of at least one of SEQ ID NOS:201-210, 254-271, 342, and 344.In particular aspects, the nucleic acid comprises a sequence selectedfrom the group of SEQ ID NOS:201-210, 254-271342, and 344. Such anucleic acid encodes a recombinant C15/full length prM/full length Epolypeptide of the invention and is generally termed a recombinantC15/full prM/full E polypeptide-encoding nucleic acid. Such nucleicacids having one or more properties of the recombinant nucleic acids aredescribed below.

[0323] The invention also provides nucleic acids that hybridize with anyof the disclosed and/or above-described nucleic acid sequences of theinvention under at least moderately stringent hybridization conditions,at least stringent hybridization conditions, at least highly stringenthybridization conditions, or preferably very stringent hybridizationconditions over substantially the entire length of a nucleic acid.“Substantially the entire length of a nucleic acid sequence” refers toat least about 50%, generally at least about 60%, at least about 70%, orat least about 75%, usually at least about 80%, at least about 85%, atleast about 88%, and typically at least about 90%, e.g., at least about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, about 99.5%, or more, of the length of the nucleicacid sequence. Thus, the invention provides a polynucleotide thatcomprises a nucleic acid sequence (a test sequence) that hybridizes toat least about 50%, preferably at least about 65%, and more preferablyat least about 80% of a reference sequence (e.g., a nucleic acidsequence disclosed herein, such as, for example, a sequence selectedfrom the group of SEQ ID NOS:156-218, 235, 254-271, 285-330, 342, and344,). More preferably, the hybridizing nucleic acid hybridizes to thedisclosed nucleic acid sequence (e.g., a sequence selected from saidabove-referenced SEQ ID NOS) under at least stringent conditions, and,even more preferably under at least high stringency conditions.Moderately stringent, stringent, and highly stringent hybridizationconditions for nucleic acid hybridization experiments are known in theart. As such, only examples of the factors that can be combined toachieve such levels of stringency are briefly discussed herein.

[0324] Exemplary moderate stringency conditions include overnightincubation at 37° C. in a solution comprising 20% formalin (orformamide), 0.5×SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.,or substantially similar conditions, e.g., the moderately stringentconditions described in Sambrook et al., supra, and/or Ausubel, supra.

[0325] Exemplary stringent (or regular stringency) conditions foranalysis of nucleic acids comprising at least 100 nucleotides includeincubation in a solution comprising 50% formalin (or formamide) with 1mg of heparin at 42° C., with the hybridization being carried outovernight. A regular stringency wash can be carried out using, e.g., asolution comprising 0.2×SSC wash at about 65° C. for about 15 minutes(see Sambrook, supra, for a description of SSC buffer). Often, theregular stringency wash is preceded by a low stringency wash to removebackground probe signal. A low stringency wash can be carried out in,for example, a solution comprising 2×SSC at about 40° C. for about 15minutes. A highly stringent wash can be carried out using a solutioncomprising 0.15 M NaCl at about 72° C. for about 15 minutes. An examplemedium (regular) stringency wash, less stringent than the regularstringency wash described above, for a duplex of, e.g., more than 1.00nucleotides, can be carried out in a solution comprising 1×SSC at 45° C.for 15 minutes. An example low stringency wash for a duplex of, e.g.,more than 100 nucleotides, is carried out in a solution of 4-6×SSC at40° C. for 15 minutes. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1.0 M Na⁺ ion, typically about 0.01 to 1.0 M Na⁺ ionconcentration (or other salts) at pH 7.0 to 8.3, and the temperature istypically at least about 30° C. Stringent conditions can also beachieved with the addition of destabilizing agents such as formamide.

[0326] High stringency conditions are conditions that use, for example,(1) low ionic strength and high temperature for washing, such as 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate(SDS) at 50° C., (2) employ a denaturing agent during hybridization,such as formamide, e.g., 50% (v/v) formamide with 0.1% BSA/0.1%Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodium phosphate buffer atpH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C., or(3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate),50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/mL), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at (i) 42° C. in 0.2×SSC,(ii) at 55° C. in 50% formamide and (iii) at 55° C. in 0.1×SSC(preferably in combination with EDTA).

[0327] More generally or alternatively, high stringency conditions areselected such that hybridization occurs at about 5° C. or less than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the test sequence hybridizes to aperfectly matched probe. In other words, the T_(m) indicates thetemperature at which the nucleic acid duplex is 50% denatured under thegiven conditions and its represents a direct measure of the stability ofthe nucleic acid hybrid. Thus, the T_(m) corresponds to the temperaturecorresponding to the midpoint in transition from helix to random coil;it depends on length, nucleotide composition, and ionic strength forlong stretches of nucleotides. Typically, under “stringent conditions,”a probe will hybridize to its target subsequence, but to no othersequences. “Very stringent conditions” are selected to be equal to theT_(m) for a particular probe.

[0328] The T_(m) of a DNA-DNA duplex can be estimated using equation(1): T_(m) (° C.)=81.5° C.+16.6 (log₁₀ M)+0.41 (%G+C)−0.72 (%f)−500/n,where M is the molarity of the monovalent cations (usually Na⁺), (%G+C)is the percentage of guanosine (G) and cytosine (C) nucleotides, (%f) isthe percentage of formalize and n is the number of nucleotide bases(i.e., length) of the hybrid. See Rapley and Walker, MOLECULARBIOMETHODS HANDBOOK supra. The T_(m) of an RNA-DNA duplex can beestimated using equation (2): T_(m) (° C.)=79.8° C.+18.5 (log₁₀ M)+0.58(%G+C)−11.8(%G+C)²−0.56 (%f)−820/n, where M is the molarity of themonovalent cations (usually Na⁺), (%G+C) is the percentage of guanosine(G) and cytosine (C) nucleotides, (%f) is the percentage of formamideand n is the number of nucleotide bases (i.e., length) of the hybrid.Id. Equations 1 and 2 above are typically accurate only for hybridduplexes longer than about 100-200 nucleotides. Id. The Tm of nucleicacid sequences shorter than 50 nucleotides can be calculated as follows:T_(m) (° C.)=4(G+C)+2(A+T), where A (adenine), C, T (thymine), and G arethe numbers of the corresponding nucleotides.

[0329] In general, unhybridized nucleic acid material desirably isremoved by a series of washes, the stringency of which can be adjusteddepending upon the desired results, in conducting hybridizationanalysis. Low stringency washing conditions (e.g., using higher salt andlower temperature) increase sensitivity, but can product nonspecifichybridization signals and high background signals. Higher stringencyconditions (e.g., using lower salt and higher temperature that is closerto the hybridization temperature) lower the background signal, typicallywith only the specific signal remaining. Addition useful guidanceconcerning such hybridization techniques is provided in, e.g., Rapleyand Walker, MOLECULAR BIOMETHODS HANDBOOK, supra (in particular, withrespect to such hybridization experiments, part I, chapter 2, “Overviewof principles of hybridization and the strategy of nucleic acid probeassays”), Elsevier, New York, as well as in Ausubel, supra, Sambrook etal., supra, Watson et al., supra, Hames and Higgins (1995) GENE PROBES1, IRL Press at Oxford University Press, Oxford, England, and Hames andHiggins (1995) GENE PROBES 2, IRL Press at Oxford University Press,Oxford, England.

[0330] Preferably, the hybridization analysis is carried out underhybridization conditions selected such that a perfectly complementaryoligonucleotide to the recombinant dengue antigen-encoding sequence orotherwise disclosed sequence hybridizes with the recombinant dengueantigen-encoding sequence with at least about 2 times (2×) (e.g., about2.5 times), desirably at least about 5 times, preferably at least about7 times, and more preferably at least about 10 times, highersignal-to-noise ratio than for hybridization of the perfectlycomplementary oligonucleotide to a control nucleic acid comprising apolynucleotide sequence that is at least about 90% identical to apolynucleotide sequence that encodes approximately the same length WTdengue virus polypeptide or encodes a corresponding WT dengue viruspolypeptide. For example, if the recombinant sequence is aPRM15/tE-encoding polynucleotide sequence, a control nucleic acid may bea wild-type PRM15/tE-encoding polynucleotide that encodes a DEN-1PRM15/tE, DEN-2 PRM15/tE, DEN-3 PRM15/tE, or DEN-4 PRM15/tE polypeptideor the control nucleic acid may comprises a polynucleotide sequence thatis at least about 90% identity with the nucleotide sequence of any of:SEQ ID NO:231 (DEN-1 PRM15/E trunc WT cDNA sequence comprising nucleicacid residues 894-2285 of GenBank Ace. No. AB074761); SEQ ID NO:232(DEN-2 PRM15/E trunc WT cDNA sequence comprising nucleic acid residues892-2274 of GenBank Acc. No. NC_(—)001474); SEQ ID NO:233 (DEN-3 PRM15/Etrunc WT cDNA sequence comprising nucleic acid residues 893-2263 ofGenBank Acc. No. M25277); and SEQ ID NO:234 (DEN-4 PRM15/E trunc WT cDNAsequence comprising nucleic acid residues 894-2285 of GenBank Acc. No.M14931). Such conditions can be considered indicative for specifichybridization.

[0331] The above-described hybridization conditions can be adjusted, oralternative hybridization conditions selected, to achieve any desiredlevel of stringency in selection of a hybridizing nucleic acid sequence.For example, the above-described highly stringent hybridization and washconditions can be gradually increased (e.g., by increasing temperature,decreasing salt concentration, increasing detergent concentration and/orincreasing the concentration of organic solvents, such as formalin, inthe hybridization or wash), until a selected set of criteria are met.For example, the hybridization and wash conditions can be graduallyincreased until a desired probe, binds to a perfectly matchedcomplementary target, with a signal-to-noise ratio that is at leastabout 2.5×, and optionally at least about 5× (e.g., about 10×, about20×, about 50×, about 100×, or even about 500×), as high as thesignal-to-noise ration observed from hybridization of the probe to anucleic acid not of the invention, such as a known nucleic acid sequenceselected from GenBank, and/or a wild-type dengue virus nucleic acidsequence, including, e.g., a wild-type dengue virus nucleic acidencoding a wild-type dengue virus truncated E polypeptide, full Epolypeptide, PRM15/tE polypeptide, PRM15/full E polypeptide, or C15/fullprM/full E polypeptide. In hybridization analyses, a recombinantpolypeptide of the invention is typically compared with or analyzed inview of a wild-type polypeptide of approximately the same length orcomprising the same or similar format (e.g., a recombinant C15/fullprM/full E polypeptide of the invention is compared with a wild-typeflavivirus C15/full prM/full E polypeptide).

[0332] In one aspect, the invention provides a nucleic acid comprising asequence of at least about 900 nucleotides (including, e.g., at leastabout 900-3000 nucleotides, at least about 1000-2000 nucleotides),preferably at least about 1200 nucleotides, and more preferably at leastabout 1300 nucleotides, that encodes an amino acid sequence that inducesan immune response to a dengue virus in a subject, wherein the nucleicacid selectively hybridizes under at least moderately stringentconditions to at least one of SEQ ID NOS:156-200 and 235 as compared toa wild-type PRM15/tE-encoding sequence (e.g., SEQ ID NOS:231-234) or aportion or fragment thereof. In one aspect, for example, the nucleicacid of the invention preferably hybridizes to at least one of SEQ IDNOS:156-200 and 235 as compared to a WT PRM15/truncated E-encodingsequence from DEN-3 (e.g., nucleotides 893-2263 of the nucleotidesequence recorded under GenBank Accession No. M25277). Preferably, thenucleic acid selectively hybridizes to at least one of SEQ IDNOS:156-200 and 235, as compared to such WT PRM15/tE-encoding sequencesand/or PRM15/tE-encoding sequences of any known wild-type and/ormodified (e.g., attenuated strain) virus, including flaviviruses (anddengue viruses) shown in GenBank. The determination of those segments ofwild-type flaviviral nucleotide sequences or WT flaviviral genomes thatencode, e.g., a WT PRM15/truncated E protein, WT PRM15/full E protein,WT C15/full prM/full E protein is within the abilities of one ofordinary skill in the art.

[0333] In another aspect, the invention provides a nucleic acidcomprising a sequence of at least about 900 nucleotides, usually atleast about 1000 nucleotides, typically at least about 1100 or about1200 nucleotides, and more preferably at least about 1300 nucleotides,that encodes an amino acid sequence that induces an immune response to adengue virus in a subject, wherein the nucleic acid selectivelyhybridizes under moderately stringent conditions to at least one of SEQID NOS:211-218 as compared to any of SEQ ID NOS:231-234 or another knownPRM15/tE-encoding dengue virus polynucleotide. Desirably, such a nucleicacid of the invention hybridizes to at least one of SEQ ID NOS:211-218more selectively than it hybridizes to any of SEQ ID NOS:231-234 oranother known PRM15/tE-encoding dengue virus polynucleotide under highlystringent conditions, preferably under regularly stringent conditions,and, most preferably, under moderately stringent conditions.

[0334] The invention also provides nucleic acids comprising a sequencethat does not hybridize to one of the specifically disclosed nucleicacid sequences of the invention (e.g., to a sequence selected from thegroup of SEQ ID NOS:156-218, 235, 253-271, 285-330, 342, and 344,), butwould so hybridize but for the degeneracy of the nucleic acid codeand/or the imposition of non-coding sequences (e.g., nucleotidesequences that are spliced out of a DNA sequence to form an RNAintermediate that encodes a polypeptide having an amino acid sequencesubstantially identical (e.g., having at least about 75%, 80%, 85%, 86%,87%, 88% or 89%, preferably at least about 90%, 91%, 92%, 93%, or 94%,and more preferably at least about 95% (e.g., about 87-95%), 96% 97%,98%, 99%, 99.5% sequence identity) with that of any recombinantpolypeptide sequence of the invention) that do not impact on the abilityof the otherwise hybridizing nucleic acid (target or test nucleic acid)to express a polypeptide that is structurally, functionally, orotherwise similar to at least one of the recombinant polypeptides of theinvention. Such non-hybridizing, but related, target sequences desirablyare at least about 60, desirably at least about 300, preferably at leastabout 900, more preferably at least about 1200, and more preferably atleast about 1300 nucleotides in length.

[0335] In one aspect, the invention provides a nucleic acid that encodesa polypeptide comprising an immunogenic amino acid sequence of at leastabout 400 amino acid residues that has substantial identity (e.g., atleast about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity) withat least one of SEQ ID NOS:1-49 and 153-155. In another aspect, theinvention includes a the nucleic acid that encodes a polypeptidecomprising an immunogenic amino acid sequence of at least about 450amino acid residues that exhibits substantial identity (e.g., at leastabout 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at least about90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity) to atleast one sequence selected from the group of SEQ ID NOS:65-116. Moreparticularly, the nucleic acid encodes a polypeptide comprising animmunogenic amino acid sequence of at least about 400 amino acidresidues that has substantial identity (e.g., at least about 75%, 80%,85%, 86%, 87%, 88% or 89%, preferably at least about 90%, 91%, 92%, 93%,or 94%, and more preferably at least about 95% (e.g., about 87-95%), 96%97%, 98%, 99%, 99.5% sequence identity) with at least one sequenceselected from any of SEQ ID NOS:66, 67, 69, 89, 93, and 108-110.

[0336] In another aspect, the invention provides a nucleic acid thatencodes a polypeptide that comprises an immunogenic amino acid sequenceof at least about 650 amino acid residues that exhibits substantialidentity (e.g., at least about 75%, 80%, 85%, 86%, 87%, 88% or 89%,preferably at least about 90%, 91%, 92%, 93%, or 94%, and morepreferably at least about 95% (e.g., about 87-95%), 96% 97%, 98%, 99%,99.5% sequence identity) with at least one of SEQ ID NOS:139-148,236-253, 343, and 345. More particularly, the nucleic acid encodes apolypeptide comprising an immunogenic amino acid sequence of at leastabout 650 amino acid residues that has substantial identity (e.g., atleast about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity) withany of SEQ ID NOS:140-141. Preferably, the nucleic acid encodes apolypeptide comprising SEQ ID NO:141 or 140.

[0337] The nucleic acids of the invention desirably encode a polypeptidethat induces an immune response to at least one dengue virus of at leastone serotype in a subject. More preferably, the nucleic acid comprises asequence that encodes at least one polypeptide comprising an immunogenicamino acid sequence that induces an immune response to at least onedengue virus of each of four virus serotypes in a subject. Desirably,the amino acid encoded by such a nucleic acid sequence induces an immuneresponse against at least one dengue virus of at least one serotype in asubject that is at least equal to or greater than the immune responseinduced against at least one dengue virus of at least one serotype inthe subject by a corresponding wild-type dengue virus polypeptide of aparticular serotype. Desirably, the nucleic acid encodes a polypeptidethat exhibits a humoral and cellular immune response to a dengue virus,preferably to one or more dengue viruses of multiple (optimally allfour) virus serotypes in a subject (e.g., a recombinant C15/fullprM/full E polypeptide, such as, e.g., a polypeptide comprising thesequence of SEQ ID NO:141), where at least the humoral, the cellular, orpreferably both immune responses promoted/induced by the encodedpolypeptide are about equal to or greater than the corresponding humoralresponse, cellular response, and/or both such immune responses,respectively, that are induced/promoted by a corresponding wild-typedengue virus polypeptide (e.g., wild-type dengue virus C15/full prM/fullE polypeptide of one or more serotypes).

[0338] In one aspect, the recombinant nucleic acid comprises apolynucleotide sequence that encodes at least one polypeptide thatinduces the production of one or more neutralizing antibodies in asubject against at least one dengue virus of at least one serotype. In aparticular aspect, the recombinant nucleic acid encodes a polypeptidethat induces the production of neutralizing antibodies against at leastone dengue virus of each of at least two, at least three, or preferablyat least four serotypes in a subject. Preferably, the nucleic acidencodes a polypeptide that induces a titer of neutralizing antibodiesagainst at least one dengue virus of at least one serotype that is atleast equal to or greater than the titer of neutralizing antibodiesinduced against the at least one dengue virus of the at least oneserotype by a wild-type nucleic acid encoding a wild-type dengue virusantigen of the same or similar size and of the same format. For example,in one aspect, a PRM15/tE polypeptide-encoding nucleic acid (e.g., SEQID NOS:156-200 and 235) is provided that encodes a polypeptide thatinduces a titer of neutralizing antibodies against at least one denguevirus of at least one serotype that is at least equal to or greater thanthe titer of neutralizing antibodies induced against the at least onedengue virus of the at least one serotype by a wild-type PRM15/tEpolypeptide-encoding nucleic acid (e.g., any of SEQ ID NOS:149-152).

[0339] Preferably, a recombinant nucleic acid of the invention comprisesa polynucleotide sequence that encodes an immunogenic polypeptide (e.g.,a nucleic acid encoding a recombinant tE, full E, PRM15/tE, C15/fullprM/full E, PRM15/full E, or prM/full E polypeptide) that induces theproduction of one or more neutralizing antibodies to at least one denguevirus of each of the four serotypes in a subject. The neutralizingantibody response induced by such encoded immunogenic polypeptidetypically does not induce ADE in a mammal upon infection of the mammalwith a dengue virus and/or upon secondary infection of the mammal with adengue virus of a different serotype than the serotype of the virus themammal was infected with before receiving the recombinant nucleic acid.

[0340] In another aspect, a recombinant nucleic acid of the inventioncomprises a polynucleotide sequence that encodes a polypeptide thatinduces a protective immune response to at least one dengue virus of atleast one serotype in a subject. In another aspect, the nucleic acidcomprises a polynucleotide sequence that encodes a polypeptide thatinduces a protective immune response against at least one dengue virusof each of at least two, preferably at least three, and more preferablyagainst all four virus serotypes when the polypeptide is expressed in asubject.

[0341] The invention further provides a nucleic acid comprising afragment of the one of the nucleic acids of the invention that encodes apolypeptide that induces an immune response against at least one denguevirus in a subject, which fragment is unique as compared to WT denguevirus antigens of similar size and/or format. Preferably, the nucleicacid fragment encodes a polypeptide that induces an immune response toat least one dengue virus of all four serotypes, and, more preferably,induces production of a neutralizing antibody and/or protective immuneresponse against at least one dengue virus of all four serotypes, in asubject (most desirably without the occurrence of ADE).

[0342] The nucleic acid of the invention can comprise any suitablenumber of other sequences in addition to the above-described recombinantdengue antigen-encoding sequences and also or alternatively can compriseany suitable number of recombinant dengue antigen-encoding sequences.For example, a nucleic acid can comprise two or more copies of a dengueantigen-encoding sequence and/or nucleotide sequences encoding multipledifferent dengue antigen-encoding sequences. For example, a nucleic acidcan comprise one or more of the following: (1) a nucleotide sequenceencoding a recombinant C15/full prM/full E polypeptide; (2) a nucleotidesequence encoding a recombinant full prM/full E polypeptide; (3) anucleotide sequence encoding a recombinant PRM15/tE polypeptide; (4) anucleotide sequence encoding a WT or recombinant signal peptide, C15 orPRM15; (5) a nucleotide sequence encoding a recombinant tE polypeptide;and (6) a nucleotide sequence encoding a recombinant full E polypeptide.

[0343] In one particular aspect, the nucleic acid comprises a secondsequence encoding an adjuvant and/or a cytokine, a costimulatorymolecule (e.g., a mammalian B7-1 or B7-2 or an amino acid sequence thathas at least substantial identity thereto or comprises a variantthereof), or a heterologous antigen (e.g., a yellow fever antigen, amalaria vaccine, etc.). The nucleic acid can comprise any suitablenumber and copy of such sequences, in any suitable combination, alongwith the recombinant dengue antigen-encoding sequence(s). The sequencescan be part of a single expression cassette, but more typically andpreferably are contained in separate expression cassettes (examples ofwhich are discussed further below). In some aspects, the recombinantdengue antigen-encoding sequence and the secondary nucleic acid sequence(e.g., the cytokine-encoding sequence) are operably linked to separateand different expression control sequences, such that they are expressedat different times and/or in response to different conditions (e.g., inresponse to different inducers).

[0344] In general, any of the nucleic acids of the invention can bemodified to increase expression in a particular host, using thetechniques exemplified herein with respect to the above-described denguevirus prM/E fusion protein-encoding sequences. Codons that are utilizedmost often in a particular host are called optimal codons, and those notutilized very often are classified as rare or low-usage codons (see,e.g., Zhang, S. P. et al. (1991) Gene 105:61-72). Codons can besubstituted to reflect the preferred codon usage of the host, a processcalled “codon optimization” or “controlling for species codon bias.”Optimized coding sequence comprising codons preferred by a particularprokaryotic or eukaryotic host can be used to increase the rate oftranslation or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, as compared with transcriptsproduced from a non-optimized sequence. Techniques for producing codonoptimized sequences are known (see, e.g., E. et al. (1989) Nuc Acids Res17:477-508). Translation stop codons can also be modified to reflecthost preference. For example, preferred stop codons for S. cerevisiaeand mammals are UAA and UGA respectively. The preferred stop codon formonocotyledonous plants is UGA, whereas insects and E. coli prefer touse UAA as the stop codon (see, e.g., Dalphin, M. E. et al. (1996) NucAcids Res 24:216-218). The arrangement of codons in context to othercodons also can influence biological properties of a nucleic acidsequences, and modifications of nucleic acids to provide a codon contextarrangement common for a particular host also is contemplated by theinventors. Thus, a nucleic acid sequence of the invention can comprise acodon optimized nucleotide sequence, i.e., codon frequency optimizedand/or codon pair (i.e., codon context) optimized for a particularspecies (e.g., the polypeptide can be expressed from a polynucleotidesequence optimized for expression in humans by replacement of “rare”human codons based on codon frequency, or codon context, such as byusing techniques such as those described in Buckingham et al. (1994)Biochimie 76(5):351-54 and U.S. Pat. Nos. 5,082,767, 5,786,464, and6,114,148). An exemplary technique for producing codon optimizedsequences is provided in Example 1.

[0345] In addition to the above-described codon optimized nucleic acidsequences (e.g., recombinant tE-encoding polynucleotide sequence, fullE-encoding polynucleotide sequence, PRM15/tE-encoding polynucleotidesequence, C15/fill length prM/full length E-encoding polynucleotidesequence, etc.), the nucleic acids of the invention generally expresspolypeptides at expression levels higher than does a correspondingwild-type polynucleotide sequence encoding a wild-type dengue viruspolypeptide sequences (e.g., WT dengue virus tE-encoding polynucleotidesequence, WT dengue virus full E-encoding polynucleotide sequence, WTdengue PRM15/tE-encoding polynucleotide sequence, WT C15/full lengthprM/full length E-encoding polynucleotide sequence, etc.). Thus, forexample, the invention provides nucleic acids encoding one or morerecombinant PRM15/tE polypeptides of the invention, wherein at least onesuch recombinant polypeptide is expressed more efficiently than anucleic acid comprising at least a portion of any one of SEQ IDNOS:231-234, of substantially the same length, when expressed from asubstantially identical expression cassette in a subject host, such as amammalian host.

[0346] Remarkably, some recombinant C15/full prM/full E polypeptides ofthe invention, expressed from the C15/full prM/full Epolypeptide-encoding polynucleotides described herein, also exhibithigher levels of secretion than codon optimized C15/full prM/full Epolypeptide-encoding sequences (e.g., any of SEQ ID NOS:215-218).

[0347] The nucleic acid is typically a DNA, and usually a doublestranded DNA sequence. However, the invention also provides singlestranded DNA, single stranded RNA, double stranded RNA, and hybridDNA/RNA nucleic acids comprising the nucleic acid sequences of theinvention also are provided. In one aspect, the invention includes a RNAsequence comprising any DNA nucleotide sequence of the inventiondescribed herein and throughout in which the thymine nucleotides in thesequence are replaced with uracil nucleotides. The invention alsoprovides, for example, an RNA nucleic acid comprising a sequence havingsubstantially identity (e.g., having at least about 75%, 80%, 85%, 90%,95% or more nucleic sequence identity) with at least one sequenceselected from the group of SEQ ID NOS:156-218, 235, 254-271, 285-330,342, and 344,). Also provided is a RNA nucleic acid comprising a DNAsequence selected from any of this group of sequences in which all ofthe thymine nucleotides in the DNA sequence are replaced with uracilnucleotides, and RNA polynucleotide sequences complementary to all suchRNA nucleic acids.

[0348] The invention further provides a RNA nucleic acid that exhibitssubstantial identity with a sequence having substantial identity (e.g.,at least about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity) withat least one selected from the group of SEQ ID NOS:285-330. Moreparticularly, the invention provides a RNA nucleic acid comprising a DNAsequence selected from any of SEQ ID NOS:285-330 in which each thymineresidue in the DNA sequence is replaced with a uracil residue. Such RNAnucleic acids typically are at least about 1000 nucleotides, typicallyabout 1200 nucleotides, and typically about 2000 nucleotides in length.The invention also provides at least one fragment of such an RNA nucleicacid that encodes an immunogenic amino acid sequence of the invention,and RNA polynucleotide sequences that are complementary to suchfragments. Also included is an RNA polynucleotide sequence thathybridizes to such an RNA nucleic acid (e.g., comprising a DNA sequenceof any of SEQ ID NOS:285-330 in which each thymine nucleotides in thesequence is replaced with uracil nucleotide) under at least moderatelystringent, preferably at least regularly stringent, and more preferablyat least highly stringent hybridization conditions.

[0349] In one aspect of the invention, the invention provides a DNAnucleic acid that comprises at least one expression control sequenceassociated with and/or typically operably linked to a recombinantnucleic acid sequence of the invention (e.g., the recombinantantigen-encoding sequence). An “expression control sequence” is anynucleic acid sequence that promotes, enhances, or controls expression(typically and preferably transcription) of another nucleic acidsequence. Suitable expression control sequences include constitutivepromoters, inducible promoters, repressible promoters, and enhancers.

[0350] Promoters exert a particularly important impact on the level ofrecombinant polypeptide expression. The nucleic acid of the invention(e.g., recombinant DNA nucleic acid) can comprise any suitable promoter.Examples of suitable promoters include the cytomegalovirus (CMV)promoter, the HIV long terminal repeat promoter, the phosphoglyceratekinase (PGK) promoter, Rous sarcoma virus (RSV) promoters, such as RSVlong terminal repeat (LTR) promoters, mouse mammary tumor virus (MMTV)promoters, HSV promoters, such as the Lap2 promoter or the herpesthymidine kinase promoter (as described in, e.g., Wagner et al. (1981)Proc Natl Acad Sci 78:144-145), promoters derived from SV40 or EpsteinBarr virus, adeno-associated viral (AAV) promoters, such as the p5promoter, metallothionein promoters (e.g., the sheep metallothioneinpromoter or the mouse metallothionein promoter (see, e.g., Palmiter etal. (1983) Science 222:809-814), the human ubiquitin C promoter, E. colipromoters, such as the lac and trp promoters, phage lambda PL promoter,and other promoters known to control expression of genes in prokaryoticor eukaryotic cells (either directly in the cell or in viruses whichinfect the cell). Promoters that exhibit strong constitutive baselineexpression in mammals, particularly humans, such as cytomegalovirus(CMV) promoters, such as the CMV immediate-early promoter (described in,for example, U.S. Pat. No. 5,168,062), and promoters having substantialsequence identity with such a promoter, are particularly preferred. Alsopreferred are recombinant promoters having novel or enhanced properties,such as those described in PCT Application Int'l Publ. No. WO 02/00897.

[0351] The promoter can have any suitable mechanism of action. Thus, thepromoter can be, for example, an “inducible” promoter, (e.g., a growthhormone promoter, metallothionein promoter, heat shock protein promoter,E1B promoter, hypoxia induced promoter, radiation inducible promoter, oradenoviral MLP promoter and tripartite leader), an inducible-repressiblepromoter, a developmental stage-related promoter (e.g., a globin genepromoter), cell-specific, or tissue specific promoter (e.g., a smoothmuscle cell α-actin promoter, myosin light-chain 1 A promoter, orvascular endothelial cadherin promoter). Suitable inducible promotersinclude ecdysone and ecdysone-analog-inducible promoters(ecdysone-analog-inducible promoters are commercially available throughStratagene (LaJolla Calif.)). Other suitable commercially availableinducible promoter systems include the inducible Tet-Off or Tet-onsystems (Clontech, Palo Alto, Calif.). The inducible promoter can be anypromoter that is up- and/or down-regulated in response to an appropriatesignal. Additional inducible promoters include arabinose-induciblepromoters, a steroid-inducible promoters (e.g., aglucocorticoid-inducible promoters), as well as pH, stress, andheat-inducible promoters.

[0352] The promoter can be, and often is, a host-native promoter, or apromoter derived from a virus that infects a particular host (e.g., ahuman beta actin promoter, human EF1α promoter, or a promoter derivedfrom a human AAV operably linked to the nucleic acid can be preferred),particularly where strict avoidance of gene expression silencing due tohost immunological reactions to sequences that are not regularly presentin the host is of concern. The polynucleotide also or alternatively caninclude a bi-directional promoter system (as described in, e.g., U.S.Pat. No. 5,017,478) linked to multiple genes of interest (e.g., multiplefusion protein encoding genes).

[0353] The nucleic acid also can be operably linked to a modified orchimeric promoter sequence. The promoter sequence is “chimeric” in thatit comprises at least two nucleic acid sequence portions obtained from,derived from, or based upon at least two different sources (e.g., twodifferent regions of an organism's genome, two different organisms, oran organism combined with a synthetic sequence). Suitable promoters alsoinclude recombinant, mutated, or recursively recombined (e.g., shuffled)promoters. Minimal promoter elements, consisting essentially of aparticular TATA-associated sequence, can, for example, be used alone orin combination with additional promoter elements. TATA-less promotersalso can be suitable in some contexts. The promoter and/or otherexpression control sequences can include one or more regulatory elementshave been deleted, modified, or inactivated. Preferred promoters includethe promoters described in Int'l Patent Application WO 02/00897, one ormore of which can be incorporated into and/or used with nucleic acidsand vectors of the invention. Other shuffled and/or recombinantpromoters also can be usefully incorporated into and used in the nucleicacids and vectors of the invention, e.g., to facilitate polypeptideexpression.

[0354] Other suitable promoters and principles related to the selection,use, and construction of suitable promoters are provided in, e.g.,Werner (1999) Mamm Genome 10(2):168-75, Walther et al. (1996) J Mol Med74(7):379-92, Novina (1996) Trends Genet 12(9):351-55, Hart (1996) SeminOncol 23(1):154-58, Gralla (1996) Curr Opin Genet Dev 6(5):526-30,Fassler et al. (1996) Methods Enzymol 273:3-29, Ayoubi et al (1996),10(4) FASEB J 10(4):453-60, Goldsteine et al. (1995) Biotechnol Annu Rev1:105-28, Azizkhan et al (1993) Crit Rev Eukaryot Gene Expr 3(4):229-54,Dynan (1989) Cell 58(1):1-4, Levine (1989) Cell 59(3):405-8, and Berk etal (1986) Annu Rev Genet 20:45-79, as well as U.S. Pat. No. 6,194,191.Other suitable promoters can be identified by use of the EukaryoticPromoter Database (release 68) (presently available athttp://www.epd.isb-sib.ch/) and other, similar, databases, such as theTranscription Regulatory Regions Database (TRRD) (version 4.1)(available at http://www.bionet.nsc.ru/trrd/) and the transcriptionfactor database (TRANSFAC) (available athttp://transfac.gbf.de/TRANSFAC/index.html).

[0355] As an alternative to a promoter, particularly in RNA vectors andconstructs, the nucleic acid sequence and/or vector can comprise one ormore internal ribosome entry sites (IRESs), IRES-encoding sequences, orRNA sequence enhancers (Kozak consensus sequence analogs), such as thetobacco mosaic virus omega prime sequence.

[0356] The invention also provides a polynucleotide (or vector) thatalso or alternatively comprises an upstream activator sequence (UAS),such as a Gal4 activator sequence (as described in, e.g., U.S. Pat. No.6,133,028) or other suitable upstream regulatory sequence (as describedin, e.g., U.S. Pat. No. 6,204,060).

[0357] In addition to an immunogenic polynucleotide sequence, apolynucleotide or vector of the invention can include any otherexpression control sequences (e.g., enhancers, translation terminationsequences, initiation sequences, splicing control sequences, etc.). Thepolynucleotide may include a Kozak consensus sequence that is functionalin a mammalian cell, which can be a naturally occurring or modifiedsequence such as the modified Kozak consensus sequences described inU.S. Pat. No. 6,107,477. The nucleic acid can include specificinitiation signals that aid in efficient translation of a codingsequence and/or fragments contained in the expression vector. Thesesignals can include, e.g., the ATG initiation codon and adjacentsequences. In cases where a coding sequence, its initiation codon andupstream sequences are inserted into the appropriate expression vector,no additional translational control signals may be needed. However, incases where only coding sequence (e.g., a mature protein codingsequence), or a portion thereof, is inserted, exogenous nucleic acidtranscriptional control signals including the ATG initiation codon mustbe provided. Furthermore, the initiation codon must be in the correctreading frame to ensure transcription of the entire insert. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. The efficiency of expression canenhanced by the inclusion of enhancers appropriate to the cell system inuse (see, e.g., Scharf D. et al. (1994) Results Probl Cell Differ20:125-62; and Bittner et al. (1987) Methods in Enzymol 153:516-544 fordiscussion). Suitable enhancers include, for example, the rous sarcomavirus (RSV) enhancer and the RTE enhancers described in U.S. Pat. No.6,225,082. Initiation signals including the ATG initiation codon andadjacent sequences are desirably incorporated in the polynucleotide. Incases where a polynucleotide sequence, its initiation codon and upstreamsequences are inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only coding sequence (e.g., a mature protein codingsequence), or a portion thereof, is inserted, exogenous nucleic acidtranscriptional control signals including the ATG initiation codon areto be provided. The initiation codon must be in the correct readingframe to ensure transcription of the entire insert. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. The efficiency of expression canenhanced by the inclusion of enhancers appropriate to the cell system inuse (see, e.g., Scharf D. et al. (1994) Results Probl Cell Differ20:125-62; and Bittner et al. (1987) Meth in Enzymol 153:516-544).

[0358] A nucleic acid of the invention (e.g., DNA) may also comprise aribosome binding site for translation initiation and atranscription-terminating region. A suitable transcription-terminatingregion is, for example, a polyadenylation sequence that facilitatescleavage and polyadenylation of the RNA transcript produced from the DNAnucleic acid. Any suitable polyadenylation sequence can be used,including a synthetic optimized sequence, as well as the polyadenylationsequence of BGH (Bovine Growth Hormone), human growth hormone gene,polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), rabbitbeta globin, and the papillomaviruses, including human papillomavirusesand BPV (Bovine Papilloma Virus). Preferred polyadenylation (polyA)sequences include the SV40 (human Sarcoma Virus-40) polyadenylationsequence and the BGH polyA sequence, which is particularly preferred.Such polyA sequences are described in, e.g., Goodwin et al. (1998)Nucleic Acids Res 26(12):2891-8, Schek et al. (1992) Mol Cell Biol12(12):5386-93, and van den Hoff et al. (1993) Nucleic Acids Res21(21):4987-8. Additional principles related to selection of appropriatepolyadenylation sequences are described in, e.g., Levitt et al. (1989)Genes Dev 1989 3(7):1019-1025, Jacob et al. (1990) Crit Rev EukaryotGene Expr 1(1):49-59, Chen et al. (1995) Nucleic Acids Res23(14):2614-2620, Moreira et al. (1995) EMBO J 14(15):3809-3819,Carswell et al. (1989) Mol Cell Biol 1989 9(10):4248-4258.

[0359] The polynucleotide can further comprise site-specificrecombination sites, which can be used to modulate transcription of thepolynucleotide, as described in, e.g., U.S. Pat. Nos. 4,959,317,5,801,030 and 6,063,627, European Patent Application 0 987 326 andInternational Patent Application WO 97/09439.

[0360] The invention further provides a nucleic acid of the inventionthat further comprises one or more immunostimulatory oligonucleotidesequences, e.g., a sequence according to the sequence patternN₁CGN₂)_(x), wherein N₁ is, 5′ to 3′, any two purines, any purine and aguanine, or any three nucleotides; N₂ is, 5′ to 3′, any two purines, anyguanine and any purine, or any three nucleotides; and x is any numbergreater than 0 or 1. Immunomodulatory sequences are known in the art,and described in, e.g., Wagner et al. (2000) Springer Semin Immunopathol22(1-2):147-52, Van Uden et al. (2000) Springer Semin Immunopathol22(1-2):1-9, and Pisetsky (1999) Immunol Res 19(1):35-46, as well asU.S. Pat. Nos. 6,207,646, 6,194,388, 6,008,200, 6,239,116, and6,218,371. The immunostimulatory oligonucleotide sequence(s) may beunmethylated. In another aspect, the invention provides a nucleic acidthat comprises a polynucleotide sequence that encodes one or morerecombinant polypeptides of the invention and further comprises at leastone polynucleotide sequence that encodes at least one immunostimulatorysequence as described herein. Alternatively, the immunostimulatoryoligonucleotide sequence is expressed from a second polynucleotidesequence that is separate from (e.g., on a separate or second vector)the first polynucleotide sequence encoding the recombinant polypeptideof the invention

[0361] In another aspect, the invention provides a nucleic acid thatcomprises a polynucleotide sequence that encodes one or more recombinantpolypeptides of the invention and further comprises at least onepolynucleotide sequence that encodes at least one protein adjuvant.Alternatively, the protein adjuvant is expressed from a secondpolynucleotide sequence that is separate from (e.g., on a separate orsecond vector) the first polynucleotide sequence encoding therecombinant polypeptide of the invention. Preferably, the adjuvant is acytokine that promotes the immune response induced by at leastimmunogenic recombinant polypeptide of the invention. Preferably, thecytokine is a granulocyte macrophage colony stimulating factor (aGM-CSF, e.g., a human GM-CSF) an interferon (e.g., human interferon(IFN) alpha, IFN-beta, IFN-gamma), or a peptide comprising an amino acidsequence that is at least substantially identical (e.g., having at leastabout 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at least about90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% or more sequenceidentity) to the sequence of at least one such cytokine. Genes encodingsuch factors are known. Human GM-CSF sequences are described in, e.g.,Wong et al. (1985) Science 228:810, Cantrell et al. (1985) Proc NatlAcad Sci 82:6250, and Kawasaki et al. (1985) Science 230:291. Desirably,in one embodiment, such a nucleic acid expresses an amount of GM-CSF ora functional analog thereof that detectably stimulates the mobilizationand differentiation of dendritic cells (DCs) and/or T-cells, increasesantigen presentation, and/or increases monocytes activity, such that theimmune response induced by the immunogenic amino acid sequence isincreased. Suitable interferon genes, such as IFN-gamma genes also areknown (see, e.g., Taya et al. (1982), Embo J 1:953-958, Cerretti et al.(1986) J Immunol 136(12): 4561, and Wang et al. (1992) Sci China B35(1):84-91). Desirably, the IFN, such as the IFN-gamma, is expressedfrom the nucleic acid in an amount that increases the immune response ofthe immunogenic amino acid sequence (e.g., by enhancing a T cell immuneresponse induced by the immunogenic amino acid sequence). AdvantageousIFN-homologs and IFN-related molecules that can be co-expressed orco-administered with the polynucleotide and/or polypeptide of theinvention are described in, e.g., International Patent Applications WO01/25438 and WO 01/36001. Co-administration (which herein includes bothsimultaneous and serial administration) of about 5 to about 10 μg of aGM-CSF-encoding plasmid with from about 5 to about 50 μg of a plasmidencoding one of the polypeptides of the invention is expected to beeffective for enhancing the antibody response in a mouse model.

[0362] In another aspect, a nucleic acid of the invention comprises a T7RNA polymerase promoter operably linked to the nucleic acid sequence,facilitating the synthesis of single stranded RNAs from the nucleic acidsequence. T7 and T7-derived sequences are known as are exemplaryexpression systems using T7 (see, e.g., Tabor and Richardson (1986) ProcNatl Acad Sci USA 82: 1074, Studier and Moffat (1986) J Mol Biol189:113, and Davanloo et al. (1964) Proc Natl Acad Sci USA 81:2035). Inone aspect, for example, nucleic acids comprising a T7 RNA polymeraseand a polynucleotide sequence encoding at least one recombinantpolypeptide of the invention are provide. Furthermore, a nucleic acid ofthe invention can comprise an origin of replication useful forpropagation in a microorganism. The bacterial origin of replication(Ori) utilized is preferably one that does not adversely affect geneexpression in mammalian cells. Examples of useful origin of replicationsequences include the f1 phage ori, RK2 oriV, pUC ori, and the pSC101ori. Preferred original of replication sequences include the ColEI oriand the p15 (available from plasmid pACYC177, New England Biolab, Inc.),alternatively another low copy ori sequence (similar to p15) can bedesirable in some contexts. The nucleic acid in this respect desirablyacts as a shuttle vector, able to replicate and/or be expressed(desirably both—such vectors capable of expression can be referred to as“expression vectors”) in both eukaryotic and prokaryotic hosts (e.g., avector comprising an origin of replication sequences recognized in botheukaryotes and prokaryotes).

[0363] A polynucleotide of the invention preferably is positioned inand/or administered in the form of a suitable delivery vehicle (i.e., avector). The vector can be any suitable vector, including chromosomal,non-chromosomal, and synthetic nucleic acid vectors (a nucleic acidsequence comprising the above described expression cassette elements(expression control and other nucleic acid associated sequences)).Examples of such vectors include derivatives of SV40, bacterialplasmids, phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, and viral nucleic acid (RNA orDNA) vectors. See, e.g., FIGS. 1 and 2.

[0364] In one aspect, the nucleic acid is be administered in a naked DNAor RNA vector, including, for example, a linear expression element (asdescribed in, e.g., Sykes and Johnston (1997) Nat Biotech 17:355-59), acompacted nucleic acid vector (as described in, e.g., U.S. Pat. No.6,077,835 and/or International Patent Application WO 00/70087), aplasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a “midge”minimal-sized nucleic acid vector (as described in, e.g., Schakowski etal. (2001) Mol Ther 3:793-800) or as a precipitated nucleic acid vectorconstruct, such as a CaPO₄ precipitated construct (as described in,e.g., International Patent Application WO 00/46147, Benvenisty andReshef (1986) Proc Natl Acad Sci USA 83:9551-55, Wigler et al. (1978),Cell 14:725, and Coraro and Pearson (1981) Somatic Cell Genetics 7:603).Nucleotide vectors and the usage thereof are known in the art (see,e.g., U.S. Pat. Nos. 5,589,466 and 5,973,972).

[0365] The vector can be an expression vector that is suitable forexpression in a bacterial system. Any vector for use in a bacterial hostcan be utilized. Suitable vectors include, for example, vectors whichdirect high level expression of fusion proteins that are readilypurified (e.g., multifunctional E. coli cloning and expression vectorssuch as BLUESCRIPT (Stratagene), pIN vectors (Van Heeke & Schuster, JBiol Chem 264:5503-5509 (1989); pET vectors (Novagen, Madison Wis.); andthe like).

[0366] The expression vector also or alternatively can be a vectorsuitable for expression in a yeast system. Any vector suitable forexpression in a yeast system can be employed. Suitable vectors for usein, e.g., Saccharomyces cerevisiae include, for example, vectorscomprising constitutive or inducible promoters such as alpha factor,alcohol oxidase and PGH (reviewed in: Ausubel, supra, Berger, supra, andGrant et al. Methods in Enzymol 153: 516-544 (1987)).

[0367] The expression vector can be propagated in a host cell. The hostcell can be a eukaryotic cell, such as a mammalian cell, a yeast cell,or a plant cell, or the host cell can be a prokaryotic cell, such as abacterial cell. Introduction of the construct into the host cell can beeffected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, electroporation, gene or vaccine gun, injection, or othercommon techniques (see, e.g., Davis et al., BASIC METHODS IN MOLECULARBIOLOGY (1986) for a description of in vivo, ex vivo, and in vitromethods).

[0368] The nucleic acids and nucleic acid vectors of the invention canfurther comprise non-naturally occurring nucleotides and nucleotidesequences. Modifications to recombinant nucleic acid sequences of theinvention can include making at least a portion or fragment of therecombinant nucleic acid sequence (e.g., a flaviviral antigen-encodingpolynucleotide sequence) comprise a phosphorothioate backbone,incorporating at least one synthetic nucleotide analog in place of or inaddition to the naturally occurring nucleotides in the nucleic acidsequence, and the addition of bases other than guanine, adenine, uracil,thymine, and cytosine, or the uses of such non-normally occurring basesin such a sequence. Such modifications can be associated with longerhalf-life, and thus can be desirable in nucleic acids vectors of theinvention. Thus, in one aspect, the invention provides recombinantnucleic acids and nucleic acid vectors comprising at least one of theaforementioned modifications, or any suitable combination thereof,wherein the nucleic acid persists longer in a mammalian host than asubstantially identical nucleic acid without such a modification ormodifications.

[0369] The expression vector can also comprises nucleotides encoding asecretion/localization sequence, which targets polypeptide expression toa desired cellular compartment, membrane, or organelle, or which directspolypeptide secretion to the periplasmic space or into the cell culturemedia. Such sequences are known in the art, and include secretion leaderor signal peptides, organelle targeting sequences (e.g., nuclearlocalization sequences, ER retention signals, mitochondrial transitsequences, chloroplast transit sequences), membrane localization/anchorsequences (e.g., stop transfer sequences, GPI anchor sequences), and thelike.

[0370] In addition, the expression vectors of the invention optionallycomprise one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells, such as dihydrofolatereductase resistance, neomycin resistance, puromycin resistance, and/orblasticidin resistance for eukaryotic cell culture, or such astetracycline or ampicillin resistance in E. coli.

[0371] Additional nucleic acids provided by the invention includecosmids. Any suitable cosmid vector can be used to replicate, transfer,and express the nucleic acid sequence of the invention. Typically, acosmid comprises a bacterial ori V, an antibiotic selection marker, acloning site, and either one or two cos sites derived from bacteriophagelambda. The cosmid can be a shuttle cosmid or mammalian cosmid,comprising a SV40 oriV and, desirably, suitable mammalian selectionmarker(s). Cosmid vectors are further described in, e.g., Hohn et al.(1988) Biotechnology 10:113-27.

[0372] The present invention also includes recombinant constructscomprising one or more of the nucleic acid sequences, including arecombinant flavivirus virus antigen-encoding polynucleotide sequence(e.g., a recombinant dengue virus antigen-encoding polynucleotidesequence), as broadly described above. The constructs comprise a vector,such as, a plasmid, a cosmid, a phage, a virus, a bacterial artificialchromosome (BAC), a yeast artificial chromosome (YAC), and the like,into which a nucleic acid sequence of the invention has been inserted,in a forward or reverse orientation.

[0373] In one aspect of the invention, delivery of a recombinant DNAsequence of the invention (e.g., a recombinant flavivirusantigen-encoding DNA sequence) is accomplished with a naked DNA plasmidor plasmid associated with one or more transfection-enhancing agents, asdiscussed further herein. The plasmid DNA vector can have any suitablecombination of features. In some aspects, preferred plasmid DNA vectorscomprise a strong promoter/enhancer region (e.g., human CMV, RSV, SV40,SL3-3, MMTV, or HIV LTR promoter), an effective poly(A) terminationsequence, an origin of replication for plasmid product in E. coli, anantibiotic resistance gene as selectable marker, and a convenientcloning site (e.g., a polylinker). A particular plasmid vector fordelivery of the nucleic acid of the invention in this respect is thevector pMaxVax10.1, the construction and features of which are describedin Example 1 (FIGS. 1 and 2). Optionally, such a plasmid vector includesat least one immunostimulatory sequence (ISS) and/or at least one geneencoding a suitable cytokine adjuvant (e.g., a GM-CSF sequence), asfurther described elsewhere herein.

[0374] In other aspects, the nucleic acid of the sequence of theinvention is positioned in and/or delivered to the host cell or hostanimal via a viral vector. Any suitable viral vector can be used in thisrespect, and several are known in the art. A viral vector can compriseany number of viral polynucleotides, alone or in combination with one ormore viral proteins, which facilitate delivery, replication, and/orexpression of the nucleic acid of the invention in a desired host cell.The viral vector can be a polynucleotide comprising all or part of aviral genome, a viral protein/nucleic acid conjugate, a virus-likeparticle (VLP), a vector similar to those described in U.S. Pat. No.5,849,586 and International Patent Application WO 97/04748, or an intactvirus particle comprising viral nucleic acids and the nucleic acid ofthe invention. A viral particle viral vector (i.e., a recombinant virus)can comprise a wild-type viral particle or a modified viral particle,particular examples of which are discussed below.

[0375] The viral vector can be a vector which requires the presence ofanother vector or wild-type virus for replication and/or expression(i.e., a helper-dependent virus), such as an adenoviral vector amplicon.Typically, such viral vectors consist essentially of a wild-type viralparticle, or a viral particle modified in its protein and/or nucleicacid content to increase transgene capacity or aid in transfectionand/or expression of the nucleic acid (examples of such vectors includethe herpes virus/AAV amplicons).

[0376] Preferably, though not necessarily, the viral vector particle isderived from, is based on, comprises, or consists of, a virus thatnormally infects animals, preferably vertebrates, such as mammals and,especially, humans. Suitable viral vector particles in this respect,include, for example, adenoviral vector particles (including any virusof or derived from a virus of the adenoviridae), adeno-associated viralvector particles (AAV vector particles) or other parvoviruses andparvoviral vector particles, papillomaviral vector particles, flaviviralvectors, alphaviral vectors, herpes viral vectors, pox virus vectors,retroviral vectors, including lentiviral vectors. Examples of suchviruses and viral vectors are in, e.g., FIELDS VIROLOGY, supra, Fieldset al., eds., VIROLOGY Raven Press, Ltd., New York (3rd ed., 1996 and4th ed., 2001), ENCYCLOPEDIA OF VIROLOGY, R. G. Webster et al., eds.,Academic Press (2nd ed., 1999), FUNDAMENTAL VIROLOGY, Fields et al.,eds., Lippincott-Raven (3rd ed., 1995), Levine, “Viruses,” ScientificAmerican Library No. 37 (1992), MEDICAL VIROLOGY, D. O. White et al.,eds., Acad. Press (2nd ed. 1994), INTRODUCTION TO MODERN VIROLOGY,Dimock, N. J. et al., eds., Blackwell Scientific Publications, Ltd.(1994).

[0377] Viral vectors that can be employed with polynucleotides of theinvention and the methods described herein include adenovirus andadeno-associated vectors, as in, e.g., Carter (1992) Curr OpinionBiotech 3:533-539 (1992) and Muzcyzka (1992) Curr Top Microbiol Immunol158:97-129 (1992). Additional types and aspects of AAV vectors aredescribed in, e.g., Buschacher et al., Blood, 5(8), 2499-504, Carter,Contrib. Microbiol 4: 85-86 (2000), Smith-Arica, Curr. Cardiol. Rep.3(1):41-49 (2001), Taj, J. Biomed. Sci. 7(4):279-91 (2000), Vigna etal., J. Gene Med. 2(5):308-16 (2000), Klimatcheva et al., Front. Biosci.4:D481-96 (1999), Lever et al., Biochem. Soc. Trans. 27(6):841-47(1999), Snyder, J. Gene Med. 1(3):166-75 (1999), Gerich et al., KneeSurg. Sports Traumatol. Arthrosc. 5(2):118-23 (1998), and During, Adv.Drug Deliv. Review 27(1):83-94 (1997), and U.S. Pat. Nos. 4,797,368,5,139,941, 5,173,414, 5,614,404, 5,658,785, 5,858,775, and 5,994,136, aswell as other references discussed elsewhere herein). Adeno-associatedviral vectors can be constructed and/or purified using the methods setforth, for example, in U.S. Pat. No. 4,797,368 and Laughlin et al., Gene23:65-73 (1983).

[0378] Another type of viral vector that can be employed withpolynucleotides and methods of the invention is a papillomaviral vector.Suitable papillomaviral vectors are known in the art and described in,e.g., Hewson (1999) Mol Med Today 5(1):8, Stephens (1987) Biochem J248(1):1-11, and U.S. Pat. No. 5,719,054. Particularly preferredpapillomaviral vectors are provided in, e.g., International PatentApplication WO 99/21979.

[0379] Alphavirus vectors can be gene delivery vectors in othercontexts. Alphavirus vectors are known in the art and described in,e.g., Carter (1992) Curr Opinion Biotech 3:533-539, Muzcyzka (1992) CurrTop Microbiol Immunol. 158:97-129, Schlesinger Expert Opin Biol Ther.2001 March; 1(2):177-91, Polo et al. Dev Biol (Basel). 2000;104:181-5,Wahlfors et al. Gene Ther. 2000 March; 7(6):472-80, Colombage et al.Virology. Oct. 10, 1998;250(1):151-63, and International PatentApplications WO 01/81609, WO 00/39318, WO 01/81553, WO 95/07994, and WO92/10578.

[0380] Another advantageous group of viral vectors are the herpes viralvectors. Examples of herpes viral vectors are described in, e.g.,Lachmann et al., Curr Opin Mol Ther 1999 October; 1(5):622-32, Fraefelet al., Adv Virus Res. 2000;55:425-51, Huard et al., Neuromuscul Disord.1997 July; 7(5):299-313, Glorioso et al., Annu Rev Microbiol.1995;49:675-710, Latchman, Mol Biotechnol. 1994 October; 2(2):179-95,and Frenkel et al., Gene Ther. 1994;1 Suppl 1:S40-6, as well as U.S.Pat. Nos. 6,261,552 and 5,599,691.

[0381] Retroviral vectors, including lentiviral vectors, also can beadvantageous gene delivery vehicles in particular contexts. There arenumerous retroviral vectors known in the art. Examples of retroviralvectors are described in, e.g., Miller, Curr Top Microbiol Immunol(1992) 158:1-24; Salmons and Gunzburg (1993) Human Gene Therapy4:129-141; Miller et al. (1994) Methods in Enzymology 217:581-599, Weberet al., Curr Opin Mol Ther. October 2001; 3(5):439-53, Hu et al.,Pharmacol Rev. December 2000 ; 52(4):493-511, Kim et al., Adv Virus Res.2000;55:545-63, Palu et al., Rev Med Virol. 2000 May-June;10(3):185-202, and Takeuchi et al., Adv Exp Med Biol. 2000;465:23-35, aswell as U.S. Pat. Nos. 6,326,195, 5,888,502, 5,580,766, and 5,672,510.

[0382] Adenoviral vectors also can be suitable viral vectors for genetransfer. Adenoviral vectors are well known in the art and described in,e.g., Graham et al (1995) Mol Biotechnol 33(3):207-220, Stephenson(1998) Clin Diagn Virol 10(2-3):187-94, Jacobs (1993) Clin Sci (Lond).85(2):117-22, U.S. Pat. Nos. 5,922,576, 5,965,358 and 6,168,941 andInt'l Patent Appns WO 98/22588, WO 98/56937, WO 99/15686, WO 99/54441,and WO 00/32754. Adenoviral vectors, herpes viral vectors and Sindbisviral vectors, useful in the practice of the invention, are describedin, e.g., Jolly (1994) Cancer Gene Therapy 1:51-64, Latchman (1994)Molec Biotechnol 2:179-195, and Johanning et al. (1995) Nucl Acids Res23:1495-1501.

[0383] Other suitable viral vectors include pox viral vectors. Examplesof such vectors are discussed in, e.g., Berencsi et al., J Infect Dis(2001)183(8):1171-9; Rosenwirth et al., Vaccine Feb. 8,2001;19(13-14):1661-70; Kittlesen et al., J Immunol (2000)164(8):4204-11; Brown et al. Gene Ther 2000 7(19):1680-9; Kanesa-thasanet al., Vaccine (2000) 19(4-5)483-91; Sten (2000) Drug 60(2):249-71.Vaccinia virus vectors are preferred pox virus vectors. Examples of suchvectors and uses thereof are provided in, e.g., Venugopal et al. (1994)Res Vet Sci 57(2):188-193, Moss (1994) Dev Biol Stand 82:55-63 (1994),Weisz et al. (1994) Mol Cell Biol 43:137-159, Mahr and Payne (1992)Immunobiology 184(2-3):126-146, Hruby (1990) Clin Microbiol Rev3(2):153-170, and International Patent Applications WO 92/07944, WO98/13500, and WO 89/08716.

[0384] Another aspect of the invention is a flaviviral vector comprisingat least one recombinant nucleic acid sequence of the invention (e.g., arecombinant PRM15 or C15 signal peptide, recombinant tEpolypeptide-encoding nucleic acid, full E polypeptide-encoding nucleicacid, PRM15/tE polypeptide-encoding nucleic acid, C15/full prM/full Epolypeptide-encoding nucleic acid, C15/full prM/tE polypeptide-encodingnucleic acid). The nucleic acid can be positioned in any suitableportion of the flaviviral genome. For example, the nucleic acid can beinserted into or used to replace a nucleotide sequence portion of thegenome, typically a nucleotide sequence portion that encodes a similaror equivalent polypeptide as does the nucleic acid. For example, arecombinant C15/full prM/full E polypeptide-encoding nucleic acid canreplace all or part wild-type C15/full prM/full E-encoding sequence ofthe flaviviral genome. Thus, a recombinant polypeptide of the inventioncan be positioned in a portion of the flaviviral envelope.

[0385] In this respect, one or more nucleic acids of the invention canbe incorporated into any suitable flaviviral vector. Examples ofsuitable vectors are described in, e.g., Bonaldo et al., Mem InstOswaldo Cruz. 2000;95 Suppl 1:215-23, Caufour et al. Virus Res. Nov. 5,2001;79(1-2):1-14, Guirakhoo et al. J Virol. August 2001;75(16):7290-304, Pletnev et al. Virology. Aug. 15, 2000;274(1):26-31,Guirakhoo et al. J Virol. June 2000; 74(12):5477-85, and InternationalPatent Applications WO 93/06214 and WO 01/53467. Techniques forconstructing recombinant viral vectors and/or modifying known orrecombinant viral vectors are disclosed in Sambrook (supra) and otherreferenced cited herein. Replication-deficient (RD) flaviviruses(including, e.g., RD dengue and yellow fever viruses) also can be usefulas vectors or for delivery vehicles. Included is a replication-deficientflavivirus (e.g., RD dengue or YF virus) comprising at least onepolypeptide of the invention in place of or in addition to the nativeflavivirus (e.g., dengue or YF virus) envelope protein or nativeflavivirus (e.g., dengue or YF virus) prM protein and envelope protein.Also contemplated is a replication-deficient flavivirus comprising atleast one nucleic acid of the invention in place of or in addition to anucleic acid segment of the WT flavivirus genome that encodes theflavivirus envelope protein or the flavivirus prM protein and envelopeprotein.

[0386] A dengue virus that is replication-deficient in mosquito hostsand that can be combined with the polypeptide of the invention (via thenucleic acid of the invention) to serve as a vaccine is described inInternational Patent Application WO 00/14245. The use of one or moreviruses and/or viral vectors of the Flaviviridae family of viruses or,including, e.g., but not limited to, a yellow fever virus or yellowfever virus vector (see, e.g., Guirakhoo et al., J. Virol.75(16):7290-304 (2001)) to deliver at least one nucleic acid and/or atleast one polypeptide of the invention is believed to be advantageous.

[0387] In another aspect, the invention provides a chimeric viruscomprising a virus of the Flaviviridae family of viruses (such as, e.g.,a yellow fever virus, such as, e.g., yellow fever 17D or the like) inwhich the complete E protein-encoding nucleic acid sequence(s) orfragment(s) thereof (e.g., a nucleic acid sequence encoding a tEprotein) of the virus of the Flaviviridae family (e.g., yellow fevervirus) is substituted with a corresponding recombinantE-protein-encoding nucleic acid sequence of the invention (or arecombinant tE-polypeptide-encoding nucleic acid). Such chimeric virusmay be an attenuated virus of the Flaviviridae family (e.g., anattenuated yellow fever virus). The invention also includes a chimericvirus of the Flaviviridae family of viruses (e.g., yellow fever virus)in which the E protein-encoding gene(s) of the virus of the Flaviviridaefamily (e.g., yellow fever virus) (or gene encoding a truncated Eprotein) is substituted with a recombinant E-protein-encoding nucleotide(or recombinant tE polypeptide-encoding nucleotide) of the invention.Typically, the nucleotide length of the substituted recombinantnucleotide of the invention is substantially equivalent to that of thereplaced virus nucleotide sequence. The nucleic acid sequence encodingthe truncated E protein typically comprises a nucleotide segmentcorresponding to the gene encoding the E protein minus the nucleotideresidues that encode at least about 8%, 10%, or 12% of the C-terminalamino acid residues of the E protein.

[0388] The invention also provides nucleic acids that comprise thegenome of a virus of the Flaviviridae family (e.g., yellow fever virusgenome or dengue virus genome) in which the nucleotide sequence of thegenome encoding the E protein (or a truncE protein) is replaced with arecombinant nucleotide sequence of the invention that encodes arecombinant full length E protein or recombinant truncE polypeptide.Included are substituted nucleic acids (isolated from the virus) andpolypeptides encoded by all such nucleic acids.

[0389] Similarly, the invention provides a chimeric virus comprising avirus of the Flaviviridae family of viruses (e.g., a yellow fever virus,such as yellow fever 17D, or dengue virus, such as DEN-2, DEN-3) inwhich a PRM15/truncated E polypeptide-encoding nucleic acid sequence ofthe virus of the Flaviviridae family (e.g., yellow fever virus) issubstituted with a recombinant PRM15/tE polypeptide-encoding nucleicacid sequence of the invention. Also provided is a chimeric viruscomprising a virus of the Flaviviridae family of viruses (such as, e.g.,yellow fever 17D, DEN-2) in which a C15/full length prM/full lengthE-polypeptide-encoding nucleic acid sequence of the virus of theFlaviviridae family (e.g., yellow fever virus) is substituted with arecombinant C15/full length prM/full length E polypeptide-encodingnucleic acid sequence of the invention. In both such embodiments, thechimeric virus may be an attenuated virus of the Flaviviridae family(e.g., an attenuated yellow fever virus). The nucleotide length of thesubstituted recombinant nucleotide is usually substantially equivalentto that of the replaced virus nucleotide sequence. Also included aresuch substituted nucleic acids (isolated from the virus) andpolypeptides encoded by all such nucleic acids.

[0390] The invention includes chimeric replication-deficient orattenuated viruses comprising a virus of the Flaviviridae family ofviruses with one or more synthetic or recombinant polypeptides ornucleic acids of the invention. Such chimeric viruses may becomereplication-deficient or attenuated by incorporation of the one or moresynthetic or recombinant polypeptides or nucleic acids of the invention.Methods of making replication-deficient or attenuated viruses bysubstituting portions of the WT flavivirus genome with synthetic orrecombinant nucleic acids as described above are included.

[0391] Also included is a chimeric virus having a genome comprising afull length chimeric flavivirus genome comprising a nucleotide sequencecomprising at least one first nucleic acid of the invention, said atleast one first nucleic acid encoding at least one recombinant orsynthetic dengue virus structural protein of the invention, wherein saidat least one first nucleic acid is linked to at least one second nucleicacid encoding at least one non-structural protein of a secondflavivirus, wherein the second flavivirus is not a dengue virus, andwherein the chimeric flavivirus is defined as an approximately11-kilobase positive strand RNA virus having a genome that codes in oneopen reading frame for three structural proteins, capsid (C),premembrane (prM) and envelope (E), followed by seven non-structuralproteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5.

[0392] Any flavivirus can be modified by the incorporation of at leastone nucleic acid of the invention, preferably at the nucleic acid level,using standard molecular biology techniques. Typically, in such aspects,at least a portion of the particular native prM/E-encoding nucleic acidsequences of interest (e.g., native sequence encoding tE, full E,PRM15/tE, or C15/full prM/full E polypeptide) are removed andreplaced/substituted with a recombinant nucleic acid of the invention,such that a recombinant polypeptide is encoded and the flaviviruscomprises a recombinant polypeptide of the invention in place of or inaddition to its native prM/E-encoding sequence interest (e.g., nativesequence encoding tE, full E, PRM15/tE, or C15/full prM/full Epolypeptide). In one aspect, this technique is performed withnon-attenuated flaviviruses by, e.g., replacing at least one nucleotidesegment or portion of the native flavivirus genome (e.g., nativesequence encoding tE, full E, PRM15/tE, or C15/full prM/full Epolypeptide) with at least one recombinant polypeptide of the invention.Such incorporation may produce a chimeric virus that is attenuated. Inanother aspect, this technique is performed with flaviviruses that arealready inactivated or that already comprise a proven attenuated virusgenome. In some particular aspects, the attenuated flavivirus is anattenuated dengue virus, e.g., PDK 53, examples of which are describedin, e.g., Bhhamarapravati et al., Vaccine 18:44-47 (2000), Men et al.,J. Virol. 70(6):3930-37 (1996), Kanesa-thasan, Kanesa-thasan et al.,Vaccine 19:3179-88 (20001) and International Patent Applications WO00/57910, WO 00/57909, WO 00/57908, and WO 00/57904. Low pathogenicityand/or low side-effect attenuated dengue viruses derived from dengue-2virus strains may be among those used. In one aspect, a dose of about 50or about 1×10² plaque forming units (pfu) or focus forming units (ffu)to about 6×10¹⁰ pfu or to about 7×10¹⁰ pfu (e.g., about 3.5×10¹⁰ pfu toabout 4.5×10¹⁰ pfu) of such a recombinant (e.g., attenuated) flaviviralvector or virus provides an amount effective to induce an immuneresponse in a suitable subject (e.g., an animal, such as a mammal,including a human) to at least one flavivirus (e.g., dengue virus of atleast one serotype). Such a dose is administered to the subject by anyroute described herein (e.g., subcutaneous injection). In anotheraspect, a dose of about 1×10² to about 5×10⁴ pfu, about 1×10² to about1.5×10⁴ pfu, about 1×10² to about 1×10³ pfu, or about 1×10³ to about1×10⁶ pfu or about 1×10⁸ pfu of a recombinant (e.g., attenuated)flaviviral vector or virus (e.g., a recombinant attenuated dengue virus)is effective to induce an immune response to at least one flavivirus(e.g., at least one dengue virus of at least one serotype uponadministration to the subject. Alternatively, the minimum lethal dosage(MLD₅₀) equivalent to any above described pfu dosage can be administeredto the subject.

[0393] The toxicity and therapeutic efficacy of vectors or viruses thatinclude recombinant molecules of the invention are determined usingstandard pharmaceutical procedures in cell cultures or experimentalanimals. One can determine the MLD₅₀ (the minimum dose lethal to 50% ofthe population) and/or the ED₅₀ (the dose therapeutically effective in50% of the population) using procedures presented herein and thoseotherwise known in the art. See also S. Plotkin and W. Orenstein,VACCINES (W. B. Saunders Co. 1999 3d ed.) for suggested doses for knownflavivirus vaccines, including yellow fever virus 17D vaccine. Nucleicacids, polypeptides, proteins, fusion proteins, transduced cells andother formulations of the present invention can be administered in anamount determined, e.g., by the MLD₅₀ of the formulation, and theside-effects thereof at various concentrations, as applied to the massand overall health of the patient. Thus, for example, the inventionprovides a method of inducing an immune response by administering a doseequal or greater to the ED₅₀ of a pharmaceutically acceptablecomposition comprising a population of recombinant yellow fever virusparticles (e.g., 17D vaccine variants) that comprise a recombinantpolypeptide or nucleic acid of the invention. Administration can beaccomplished via single dose or divided doses (either byco-administration, serial administration, or combinations thereof).Administration techniques and protocols are described in, e.g., Plotkin(VACCINES) supra and other references cited herein. In a related sense,techniques for assessing dosage of the nucleic acid, polypeptide,vector, and cell compositions effective for inducing immunity aredescribed in, e.g., European Patent Application 1 156 333 and referencescited therein.

[0394] In some aspects, it is preferred that the virus vector isattenuated or replication-deficient in a host cell or host. AAV vectors,which are naturally replication-deficient in the absence ofcomplementing adenoviruses or at least adenovirus gene products(provided by, e.g., a helper virus, plasmid, or complementation cell),are preferred in this respect. By “replication-deficient” is meant thatthe viral vector comprises a genome that lacks at least onereplication-essential gene function. A deficiency in a gene, genefunction, or gene or genomic region, as used herein, is defined as adeletion of sufficient genetic material of the viral genome to impair orobliterate the function of the gene whose nucleic acid sequence wasdeleted in whole or in part. Replication-essential gene functions arethose gene functions that are required for replication (i.e.,propagation) of a replication-deficient viral vector. The essential genefunctions of the viral vector particle vary with the type of viralvector particle at issue. Examples of replication-deficient viral vectorparticles are described in, e.g., Marconi et al., Proc. Natl. Acad. Sci.USA, 93(21), 11319-20 (1996), Johnson and Friedmann, Methods Cell Biol.,43 (pt. A), 211-30 (1994), Timiryasova et al., J. Gene Med., 3(5),468-77 (2001), Burton et al., Stem Cells, 19(5), 358-77 (2001), Kim etal., Virology, 282(1), 154-67 (2001), Jones et al., Virology, 278(1),137-50 (2000), Gill et al., J. Med. Virol., 62(2), 127-39 (2000), Chenand Engleman, J. Virol., 74(17), 8188-93 (2000), Marconi et al., GeneTher., 6(5), 904-12 (1999), Krisky et al., Gene Ther., 5(11), 1517-30(1998), Bieniasz et al., Virology, 235(1), 65-72 (1997), Strayer et al.,Biotechniques, 22(3), 447-50 (1997), Wyatt et al., Vaccine, 14(15),1451-8 (1996), and Penciolelli et al., J. Virol., 61(2), 579-83 (1987).Other replication-deficient vectors are based on simple MuLV vectors.See, e.g., Miller et al. (1990) Mol Cell Biol 10:4239 (1990); Kolberg(1992) J NIH Res 4:43, and Cornetta et al. (1991) Hum Gene Ther 2:215).

[0395] The basic construction of recombinant viral vectors is wellunderstood in the art and involves using standard molecular biologicaltechniques such as those described in, e.g., Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL (Cold Spring Harbor Press 1989) and thethird edition thereof (2001), Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY (Wiley Interscience Publishers 1995), and Watson etal., RECOMBINANT DNA, (2d ed.), and several of the other referencesmentioned herein, all of which are incorporated herein by reference intheir entirety for all purposes. For example, adenoviral vectors can beconstructed and/or purified using the methods set forth, for example, inGraham et al., Mol. Biotechnol., 33(3), 207-220 (1995), U.S. Pat. No.5,965,358, Donthine et al., Gene Ther., 7(20), 1707-14 (2000), and otherreferences described herein. Adeno-associated viral vectors can beconstructed and/or purified using the methods set forth, for example, inU.S. Pat. No. 4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).Similar techniques are known in the art with respect to other viralvectors, particularly with respect to herpes viral vectors (see e.g.,Lachman et al., Curr. Opin. Mol. Ther., 1(5), 622-32 (1999)), lentiviralvectors, and other retroviral vectors. In general, the viral vectorcomprises an insertion of the nucleic acid (for example, a wild-typeadenoviral vector can comprise an insertion of up to 3 KB withoutdeletion), or, more typically, comprises one or more deletions of thevirus genome to accommodate insertion of the nucleic acid and additionalnucleic acids, as desired, and to prevent replication in host cells.

[0396] In one aspect, the viral vector desirably is a targeted viralvector, comprising a restricted or expanded tropism as compared to awild-type viral particle of similar type. Targeting is typicallyaccomplished by modification of capsid and/or envelope proteins of thevirus particle. Examples of targeted virus vectors and relatedprinciples are described in, e.g., International Patent Applications WO92/06180, WO 94/10323, WO 97/38723, and WO 01/28569, and WO 00/11201,Engelstadter et al., Gene Ther., 8(15), 1202-6 (2001), van Beusechem etal., Gene Ther., 7(22), 1940-6 (2000), Boerger et al., Proc. Natl. Acad.Sci. USA, 96(17), 9867-72 (1999), Bartlett et al., Nat. Biotechnol.,17(2), 181-6 (1999), Girod et al., Nat. Med., 5(9), 1052-56 (as modifiedby the erratum in Nat. Med., 5(12), 1438) (1999), J Gene Med.September-October1999; 1(5):300-11, Karavanas et al. Crit Rev OncolHematol. June 1998; 28(1):7-30, Wickham et al., J. Virol., 71(10),7663-9 (1997), Cripe et al., Cancer Res., 61(7), 2953-60 (2001), vanDeutekom et al., J. Gene Med., 1(6), 393-9 (1999), McDonald et al., J.Gene Med., 1(2), 103-10 (1999), Peng, Curr Opin Biotechnol. October1999;10(5):454-7, Staba et al., Cancer Gene Ther., 7(1), 13-9 (2000), Kibbeet al., Arch. Surg., 135(2), 191-7 (2000), Harari et al., Gene Ther.,6(5), 801-7 (2000), and Bouri et al., Hum Gene Ther., 10(10), 1633-40(1999), and Laquerre et al., J. Virol., 72(12), 9683-97 (1997), BuchholzCurr Opin Mol Ther. October1999; 1(5):613-21, U.S. Pat. Nos. 6,261,554,5,962,274, 5,695,991, and 6,251,654, and European Patent Appns 1 002 119and 1 038 967. Particular targeted vectors and techniques for producingsuch vectors are provided in Int'l Patent Appn WO 99/23107.

[0397] The viral vector particle can be a chimeric viral vectorparticle. Examples of chimeric viral vector particles are described in,e.g., Reynolds et al., Mol. Med. Today, 5(1), 25-31 (1999), Boursnell etal., Gene, 13, 311-317 (1991), Dobbe et al., Virology, 288(2), 283-94(2001), Grene et al., AIDS Res. Human. Retroviruses, 13(1), 41-51(1997), Reimann et al., J. Virol., 70(10), 6922-8 (1996), Li et al., J.Virol., 67(11), 6659-66 (1993), Dong et al., J. Virol., 66(12), 7374-82(1992), Wahlfors, Hum Gene Ther. May 1, 1999;10(7):1197-206, Reynolds etal., Mol. Med. Today, 5(1), 25-31 (1999), Boursnell et al., Gene, 13,311-317 (1991).and U.S. Pat. Nos. 5,877,011, 6,183,753, 6,146,643, and6,025,341.

[0398] Non-viral vectors of the invention also can be associated withmolecules that target the vector to a particular region in the host(e.g., a particular organ, tissue, and/or cell type). For example, anucleotide can be conjugated to a targeting protein, such as a viralprotein that binds a receptor or a protein that binds a receptor of aparticular target (e.g., by a modification of the techniques provided inWu and Wu, J. Biol. Chem., 263(29), 14621-24 (1988)). Targeted cationiclipid compositions also are known in the art (see, e.g., U.S. Pat. No.6,120,799). Other techniques for targeting genetic constructs areprovided in International Patent Application WO 99/41402.

[0399] In a further embodiment, the present invention provides hostcells comprising one or more of any of the above-described nucleicacids, vectors, polypeptides, antibodies, fusion proteins, or otherconstructs of the invention, or any combination of one or more of these.The host cell can be a eukaryotic cell, such as a mammalian cell, ayeast cell, or a plant cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, electroporation, gene or vaccine gun, injection,or other common techniques (see, e.g., Davis, L., Dibner, M., andBattey, I. (1986) BASIC METHODS IN MOLECULAR BIOLOGY) for in vivo, exvivo, and in vitro methods.

[0400] A host cell strain is optionally chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of theprotein include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing that cleaves a “pre” or a “prepro” form ofthe protein may also be important for correct insertion, folding and/orfunction. Different host cells such as E. coli, Bacillus sp., yeast ormammalian cells such as CHO, HeLa, BHK, MDCK, HEK 293, WI38, etc. havespecific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced foreign protein.

[0401] A nucleic acid of the invention can be inserted into anappropriate host cell (in culture or in a host organism) to permit thehost to express the protein. Any suitable host cell can be usedtransformed/transduced by the nucleic acids of the invention. Examplesof appropriate expression hosts include: bacterial cells, such as E.coli, Streptomyces, Bacillus sp., and Salmonella typhimurium; fungalcells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurosporacrassa; insect cells such as Drosophila and Spodoptera frugiperda;mammalian cells such as Vero cells, HeLa cells, CHO cells, COS cells,WI38 cells, N1H-3T3 cells (and other fibroblast cells, such as MRC-5cells), MDCK cells, KB cells, SW-13 cells, MCF7 cells, BHK cells,HEK-293 cells, Bowes melanoma cells, and plant cells, etc. It isunderstood that not all cells or cell lines need to be capable ofproducing fully functional polypeptides or fragments thereof; forexample, antigenic fragments of the polypeptide may be produced in abacterial or other expression system. Additional examples of suitablecells are described, for example, in U.S. Pat. No. 5,994,106 and Int'lPatent Application WO 95/34671.

[0402] The present invention also provides host cells that aretransduced, transformed or transfected with vectors of the invention. Avector of the invention typically comprises a nucleic acid of theinvention (e.g., recombinant PRM15 or C15 signal peptide-encodingnucleic acid, tE polypeptide-encoding nucleic acid, full Epolypeptide-encoding nucleic acid, PRM15/tE polypeptide-encoding nucleicacid, C15/full prM/full E-encoding nucleic acid). Host cells aregenetically engineered (e.g., transduced, transformed or transfected)with the vectors of this invention, which may be, for example, a cloningvector or an expression vector. The vector may be, for example, in theform of a plasmid, a viral particle, a phage, etc. Cells suitable fortransduction and/or infection with viral vectors of the invention forproduction of the recombinant polypeptides of the invention and/or forreplication of the viral vector of the invention include theabove-described cells.

[0403] Examples of cells that have been demonstrated as suitable forpackaging of viral vector particles are described in, e.g., Inoue etal., J. Virol., 72(9), 7024-31 (1998), Polo et al., Proc. Natl. Acad.Sci., 96(8), 4598-603 (1999), Farson et al., J. Gene Med., 1(3), 195-209(1999), Sheridan et al., Mol. Ther., 2(3), 262-75 (2000), Chen et al.,Gene Ther., 8(9), 697-703 (2001), and Pizzaro et al., Gene Ther., 8(10),737-745 (2001). For replication-deficient viral vectors, such as AAVvectors, complementing cell lines, or cell lines transformed with helperviruses, or cell lines transformed with plasmids encoding essentialgenes, are necessary for replication of the viral vector.

[0404] The engineered host cells can be cultured in conventionalnutrient media modified as appropriate for activating promoters,selecting transformants, or amplifying the gene of interest. The cultureconditions, such as temperature, pH, and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art and in the references cited herein, including,e.g., ANIMAL CELL TECHNOLOGY, Rhiel et al., eds., (Kluwer AcademicPublishers 1999), Chaubard et al., Genetic Eng. News, 20(18) (2000), Huet al., ASM News, 59, 65-68 (1993), Hu et al., Biotechnol. Prog., 1,209-215 (1985), Martin et al., Biotechnol., (1987), Freshney, CULTURE OFANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE, 4^(TH) ED., (Wiley, 2000),Mather, INTRODUCTION TO CELL AND TISSUE CULTURE: THEORY AND TECHNIQUE,(Plenum Press, 1998), Freshney, CULTURE OF IMMORTALIZED CELLS, 3^(RD)ED., (John Wiley & Sons, 1996), CELL CULTURE: ESSENTIAL TECHNIQUES,Doyle et al., eds. (John Wiley & Sons 1998), and GENERAL TECHNIQUES OFCELL CULTURE, Harrison et al., eds., (Cambridge Univ. Press 1997). Thenucleic acid also can be contained, replicated, and/or expressed inplant cells. Techniques related to the culture of plant cells aredescribed in, e.g., Payne et al. (1992) PLANT CELL AND TISSUE CULTURE INLIQUID SYSTEMS John Wiley & Sons, Inc. New York, N.Y.; Gamborg andPhillips (eds.) (1995) PLANT CELL, TISSUE AND ORGAN CULTURE: FUNDAMENTALMETHODS Springer Lab Manual, Springer-Verlag (Berlin Heidelberg N.Y.)and PLANT MOLECULAR BIOLOGY (1993) R. R. D. Croy (ed.) Bios ScientificPublishers, Oxford, U.K. ISBN 0 12 198370 6. Cell culture media ingeneral are set forth in Atlas and Parks (eds.) THE HANDBOOK OFMICROBIOLOGICAL MEDIA (1993) CRC Press, Boca Raton, Fla.

[0405] For long-term, high-yield production of recombinant proteins,stable expression can be used. For example, cell lines that stablyexpress a polypeptide of the invention are transduced using expressionvectors which comprise viral origins of replication or endogenousexpression elements and a selectable marker gene. Following theintroduction of the vector, cells may be allowed to grow for 1-2 days inan enriched media before they are switched to selective media. Thepurpose of the selectable marker is to confer resistance to selection,and its presence allows growth and recovery of cells that successfullyexpress the introduced sequences. For example, resistant clumps ofstably transformed cells can be proliferated using tissue culturetechniques appropriate to the cell type.

[0406] Host cells transformed with an expression vector and/orpolynucleotide are optionally cultured under conditions suitable for theexpression and recovery of the encoded protein from cell culture. Thepolypeptide or fragment thereof produced by such a recombinant cell maybe secreted, membrane-bound, or contained intracellularly, depending onthe sequence and/or the vector used. Expression vectors comprisingpolynucleotides encoding mature polypeptides of the invention can bedesigned with signal sequences that direct secretion of the maturepolypeptides through a prokaryotic or eukaryotic cell membrane.Principles related to such signal sequences are discussed elsewhereherein.

[0407] Cell-free transcription/translation systems can also be employedto produce recombinant polypeptides of the invention or fragmentsthereof using DNAs and/or RNAs of the present invention or fragmentsthereof. Several such systems are commercially available. A generalguide to in vitro transcription and translation protocols is found inTymms (1995) IN VITRO TRANSCRIPTION AND TRANSLATION PROTOCOLS: METHODSIN MOLECULAR BIOLOGY Volume 37, Garland Publishing, NY.

[0408] The invention further provides a composition comprising at leastone polypeptide of the invention, at least one vector of the invention,at least one nucleic acid of the invention, at least one cell of theinvention, at least one antibody of the invention, or any combinationthereof and a carrier, excipient, or diluent. Such compositions cancomprise any suitable amount of any suitable number of polypeptides,fusion proteins, nucleic acids, vectors, and/or cells of the invention.Also provided are pharmaceutical compositions comprising at least onepolypeptide, vector, nucleic acid, cell, antibody of the invention, orany combination thereof and a pharmaceutically acceptable carrier,excipient, or diluent.

[0409] For example, in one embodiment, the invention providescomposition that comprises an excipient or carrier and a plurality ofmore recombinant polypeptides of the invention (e.g., two, three, four,or more recombinant polypeptide), wherein the composition induces ahumoral and/or T cell immune response(s) against at least one flavivirusof at least one serotype (e.g., at least one dengue of at least onedengue virus serotype) in a subject, such as a mammal. Correspondingpharmaceutical compositions comprising a pharmaceutically acceptableexcipient or carrier are also provided.

[0410] In another aspect, the invention provides compositions (includingpharmaceutical compositions) that comprise an excipient or carrier (orpharmaceutically acceptable excipient or carrier) and a plurality ofmore dengue antigens (e.g., two, three, four, or more antigens), whereinat least one of the antigen is a recombinant polypeptide of theinvention and the composition induces a humoral and/or T cell immuneresponse(s) against at least one dengue virus of at least one serotypein a subject, such as a mammal. More preferably, the combined dengueantigen composition induces a protective immune response(s) against oneor more dengue viruses of at least two, three, or all four virusserotypes in a subject.

[0411] In another aspect, the invention provides a composition (orpharmaceutical composition) comprising: (1) an excipient or carrier (orpharmaceutically acceptable excipient or carrier); (2) a polynucleotidecomprising a nucleic acid sequence, that when expressed in a subject(e.g., mammal), produces a recombinant polypeptide of the invention);and (3) at least one additional nucleic acid sequence encoding an WT orrecombinant dengue virus antigen and/or at least one WT or recombinantdengue virus antigen polypeptide. Such a recombinant or WT dengue virusantigen may in the form of, e.g., a WT or recombinant tE polypeptide,full E polypeptide, PRM15/tE polypeptide, C15/full prM/full Epolypeptide, PRM15/full E polypeptide, or C15/full prM/tE polypeptide.Such composition induces a humoral and/or T cell response(s), andpreferably a protective immune response against one or more dengueviruses of multiple virus serotypes in a subject. In such compositions,the recombinant nucleic acid of the invention, which encodes arecombinant polypeptide of the invention, and the nucleic acid sequenceencoding at least one additional dengue virus antigen(s) can be in thesame polynucleotide, or located on two or more different or separatepolynucleotides, and, if desired, the various polynucleotide sequencescan be isolated or positioned in one or more suitable vectors. In suchcompositions, the at least one additional dengue antigen co-administeredand/or co-expressed with the recombinant polypeptide of the inventioncan be a recombinant polypeptide of the invention, a naturally occurringWT dengue virus antigen, or a known variant of a naturally occurringdengue virus antigen (e.g., a hybrid DEN-2/DEN-3 envelope as describedin Bielefeldt-Ohmann et al., J. Gen Virol 78(11):2723-2733 (1997)). Inone aspect, the at least one additional dengue antigen comprises atleast one naturally occurring epitope (e.g., T cell epitope), such thatthe composition induces an immune response (e.g., T cell response) thatis essentially equivalent to the immune response (e.g., T cell response)induced by a corresponding WT dengue virus antigen of the same orsimilar format (e.g., WT tE polypeptide, full E polypeptide, PRM15/tEpolypeptide, C15/full prM/full E polypeptide, PRM15/full E polypeptide,or C15/full prM/tE polypeptide). Usually, in all of the analysesdescribed throughout, for proper comparison, a recombinant dengue virusantigen is compared with WT dengue virus antigen having the same or asubstantially similar format and/or size (e.g., a recombinant PRM15/tEpolypeptide is compared with a WT PRM15/tE polypeptide). Similarly, anucleic acid encoding a recombinant dengue virus antigen of a particularformat and/or size is compared with a nucleic acid encoding a WT denguevirus antigen having the same or substantially similar format or size.

[0412] In one aspect, a composition of the invention includes apolynucleotide comprising a first nucleic acid sequence encoding apolypeptide of the invention that induces a neutralizing antibodyresponse against at least one dengue virus of each of at least twodengue virus serotypes (and preferably against one or more dengueviruses of all four serotypes) and a plurality of additional nucleicacid sequences encoding peptides comprising known virus epitopes (e.g.,T cell epitopes) from DEN-1, DEN-2, DEN-3, and/or DEN-4.

[0413] Additionally or alternatively, the composition can include one ormore polypeptides of the invention selected for the retention of atleast one known wild-type dengue epitope (e.g., T cell epitope). Suchwild-type dengue epitopes are known in the art and include, e.g., theregions of the DEN-2 envelope protein comprising from about amino acidresidues 35-50, 59-78, 135-157, 145-169, 240-250, 270-298, 295-307,335-354, and/or 356-376 (see, e.g., Rothman et al., J Virol70(10):6540-6546 (1996), Leclerc et al. Mol Immunol 30(7):613-625(1993), and Roehrig et al. Virology 191(1):31-38 (1994)). Additionally,epitopes (e.g., T cell epitopes) within dengue E and prM proteins can beidentified by epitope analysis (e.g., T cell epitope analysis) by one ofordinary skill using programs and algorithms known in the art, examplesof which are further described herein, and by subsequent sequencecomparison to identify polypeptide(s) of the invention that retain suchidentified epitopes for addition to the composition.

[0414] Desirably, a pharmaceutical composition of the inventioncomprises a pharmaceutically acceptable excipient or carrier and anantigenic or immunogenic amount of at least one recombinant polypeptide,polynucleotide, or vector of the invention (or a combination of any ofthese) sufficient to induce a immune response to at least one flavivirus(e.g., a dengue virus) of at least one serotype in a subject to whichthe pharmaceutical composition is administered in vivo or via ex vivomethods. In one particular aspect, the an amount of at least recombinantpolypeptide, polynucleotide, or vector of the invention in thepharmaceutical composition is sufficient to induce a protective immuneresponse in a subject to which the pharmaceutical composition isadministered; that is, the amount is sufficient to protect againstinfection by the at least one flavivirus of at least one serotype. Inanother aspect, the pharmaceutical composition comprises apharmaceutically acceptable excipient or carrier and an antigenic orimmunogenic amount of at least one recombinant polypeptide,polynucleotide, or vector of the invention (or a combination of any ofthese) sufficient to induce a immune response in a subject to which thecomposition is delivered against at least one dengue virus of at leasttwo, at least three, or four virus serotypes.

[0415] The composition (or pharmaceutical composition) can be anynon-toxic composition that does not interfere with the immunogenicity ofthe at least one polypeptide, polynucleotide, or vector of the inventionincluded therein. The composition can comprise one or more excipients orcarriers, and the pharmaceutical composition comprises one or morepharmaceutically acceptable carriers. A wide variety of acceptablecarriers, diluents, and excipients are known in the art. There are awide variety of suitable formulations of compositions and pharmaceuticalcompositions of the present invention. For example, a variety of aqueouscarriers can be used, e.g., buffered saline, such as phosphate-bufferedsaline (PBS), and the like are advantageous in injectable formulationsof the polypeptide, polynucleotide, and/or vector of the invention.These solutions are preferably sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may comprise pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as, e.g., pH adjusting and buffering agents, toxicityadjusting agents and the like, for example, sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. Any suitable carrier can be used in the administration of thepolynucleotide, polypeptide, and/or vector of the invention, and severalcarriers for administration of therapeutic proteins are known in theart.

[0416] The composition, pharmaceutical composition and/orpharmaceutically acceptable carrier also can include diluents, fillers,salts, buffers, detergents (e.g., a nonionic detergent, such asTween-80), stabilizers, stabilizers (e.g., sugars or protein-free aminoacids), preservants, tissue fixatives, solubilizers, and/or othermaterials suitable for inclusion in a pharmaceutically composition.Examples of suitable components of the pharmaceutical composition inthis respect are described in, e.g., Berge et al., J. Pharm. Sci.,66(1), 1-19 (1977), Wang and Hanson, J. Parenteral. Sci. Tech., 42,S4-S6 (1988), U.S. Pat. Nos. 6,165,779 and 6,225,289, and elsewhereherein. The pharmaceutical composition also can include preservatives,antioxidants, or other additives known to those of skill in the art.Additional pharmaceutically acceptable carriers are known in the art.Examples of additional suitable carriers are described in, e.g.,Urquhart et al., Lancet, 16, 367 (1980), Lieberman et al.,PHARMACEUTICAL DOSAGE FORMS—DISPERSE SYSTEMS (2nd ed., vol. 3, 1998),Ansel et al., PHARMACEUTICAL DOSAGE FORMS & DRUG DELIVERY SYSTEMS (7thed. 2000), Martindale, THE EXTRA PHARMACOPEIA (31 st edition),Remington's PHARMACEUTICAL SCIENCES (16th-20th editions), THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, Goodman and Gilman, Eds. (9thed.—1996), WILSON AND GISVOLDS TEXTBOOK OF ORGANIC MEDICINAL ANDPHARMACEUTICAL CHEMISTRY, Delgado and Remers, Eds. (10th ed.—1998), andU.S. Pat. Nos. 5,708,025 and 5,994,106. Principles of formulatingpharmaceutically acceptable compositions are described in, e.g., Platt,Clin. Lab Med., 7, 289-99 (1987), Aulton, PHARMACEUTICS: THE SCIENCE OFDOSAGE FORM DESIGN, Churchill Livingstone (New York) (1988),EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP (1998), and “DrugDosage,” J. Kans. Med. Soc., 70(1), 30-32 (1969). Additionalpharmaceutically acceptable carriers particularly suitable foradministration of vectors are described in, for example, InternationalPatent Application WO 98/32859.

[0417] The composition or pharmaceutical composition of the inventioncan comprise or be in the form of a liposome. Suitable lipids forliposomal formulation include, without limitation, monoglycerides,diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bileacids, and the like. Preparation of such liposomal formulations isdescribed in, e.g., U.S. Pat. Nos. 4,837,028 and 4,737,323.

[0418] The form of the compositions or pharmaceutical composition can bedictated, at least in part, by the route of administration of thepolypeptide, polynucleotide, cell, and/or vector of interest. Becausenumerous routes of administration are possible, the form of thepharmaceutical composition and/or components thereof can vary. Forexample, in transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are preferably included inthe composition. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. In contrast, in transmucosaladministration can be facilitated through the use of nasal sprays orsuppositories.

[0419] A common administration form for compositions, includingpharmaceutical compositions, comprising the polypeptides and/orpolynucleotides of the invention is by injection. Injectablepharmaceutically acceptable compositions comprise one or more suitableliquid carriers such as water, petroleum, physiological saline,bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.), phosphatebuffered saline (PBS), or oils. Liquid pharmaceutical compositions canfurther include physiological saline solution, dextrose (or othersaccharide solution), polyols, or glycols, such as ethylene glycol,propylene glycol, PEG, coating agents which promote proper fluidity,such as lecithin, isotonic agents, such as mannitol or sorbitol, organicesters such as ethyoleate, and absorption-delaying agents, such asaluminum monostearate and gelatins. Preferably, the injectablecomposition is in the form of a pyrogen-free, stable, aqueous solution.Preferably, the injectable aqueous solution comprises an isotonicvehicle such as sodium chloride, Ringer's injection solution, dextrose,lactated Ringer's injection solution, or an equivalent delivery vehicle(e.g., sodium chloride/dextrose injection solution). Formulationssuitable for injection by intraarticular (in the joints), intravenous,intramuscular, intradermal, subdermal, intraperitoneal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can include antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient (e.g., PBS and/or saline solutions, such as 0.1 MNaCl), and aqueous and non-aqueous sterile suspensions that can includesuspending agents, solubilizers, thickening agents, stabilizers, andpreservatives.

[0420] The administration of a polypeptide, polynucleotide, or vector ofthe invention can be facilitated by a delivery device be formed of anysuitable material. Examples of suitable matrix materials for producingnon-biodegradable administration devices include hydroxapatite,bioglass, aluminates, or other ceramics. In some applications, asequestering agent, such as carboxymethylcellulose (CMC),methylcellulose, or hydroxypropylmethylcellulose (HPMC), can be used tobind the polypeptide, polynucleotide, or vector to the device forlocalized delivery.

[0421] In another aspect, a polynucleotide or vector of the inventioncan be formulated with one or more poloxamers,polyoxyethylene/polyoxypropylene block copolymers, or other surfactantsor soap-like lipophilic substances for delivery of the polynucleotide orvector to a population of cells or tissue or skin of a subject in vivo,ex vivo, or in in vitro systems. See e.g., U.S. Pat. Nos. 6,149,922,6,086,899, and 5,990,241, each of which is incorporated herein byreference in its entirety for all purposes.

[0422] Vectors and polynucleotides of the invention can be desirablyassociated with one or more transfection-enhancing agents. In someembodiments, a nucleic acid and/or nucleic acid vector of the inventiontypically is associated with stability-promoting salts, carriers (e.g.,PEG), and/or formulations that aid in transfection (e.g., sodiumphosphate salts, dextran carriers, iron oxide carriers, or biolisticdelivery (“gene gun”) carriers, such as gold bead or powder carriers)(see, e.g., U.S. Pat. No. 4,945,050). Additional transfection-enhancingagents include viral particles to which the nucleic acid/nucleic acidvector can be conjugated, a calcium phosphate precipitating agent, aprotease, a lipase, a bipuvicaine solution, a saponin, a lipid(preferably a charged lipid), a liposome (preferably a cationicliposome, examples of which are described elsewhere herein), atransfection facilitating peptide or protein-complex (e.g., apoly(ethylenimine), polylysine, or viral protein-nucleic acid complex),a virosome, or a modified cell or cell-like structure (e.g., a fusioncell).

[0423] Nucleic acids of the invention can also be delivered by in vivoor ex vivo electroporation methods, including, e.g., those described inU.S. Pat. Nos. 6,110,161 and 6,261,281 and Widera et al., J. of Immunol.164: 4635-4640 (2000), each of which is incorporated herein by referencein its entirety for all purposes.

[0424] Transdermal administration of at least one recombinantpolypeptide, polynucleotide, and/or vector of the invention can befacilitated by a transdermal patch comprising the at least onepolypeptide, polynucleotide, and/or vector in any suitable compositionin any suitable form. Such transdermal patch devices are provided by theinvention. For example, the at least one polypeptide, polynucleotide,and/or vector can be contained in a liquid reservoir in a drug reservoirpatch device, or, alternatively, the polypeptide and/or polynucleotidecan be dispersed throughout a material suitable for incorporation in asimple monolithic transdermal patch device. Typically, the patchcomprises an immunogenic or antigenic amount of the polypeptide.Examples of such patch devices are known in the art. The patch devicecan be either a passive device or a device capable of iontophoreticdelivery of the at least polypeptide, polynucleotide, and/or vector tothe skin or tissue of the subject. Methods of promoting immunity to atleast one dengue virus of at least one serotype in a subject compriseadministering such a transdermal patch to the skin of the subject for aperiod of time and under conditions sufficient to promote immunity tothe at least one dengue virus.

[0425] The composition, particularly the pharmaceutical composition,desirably comprises an amount of at least one polynucleotide,polypeptide, and/or vector in a dose sufficient to induce a protectiveimmune response in a subject, preferably a human, upon administration.The composition can comprise any suitable dose of the at least onepolypeptide, polynucleotide, and/or vector. Proper dosage can bedetermined by any suitable technique. In a simple dosage testingregimen, low doses of the composition are administered to a test subjector system (e.g., an animal model, cell-free system, or whole cell assaysystem). Considerations in dosing for immunogenic polypeptide,polynucleotide, and/or vector compositions (as well as for gene transferby viral vectors) are known in the art. Briefly, dosage is commonlydetermined by the efficacy of the particular nucleic acid, polypeptide,and/or vector, the condition of the patient, as well as the body weightand/or target area of the patient to be treated. The size of the dose isalso determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of any such particularpolypeptide, nucleic acid, vector, formulation, composition, transducedcell, cell type, or the like in a particular patient. Principles relatedto dosage of therapeutic and prophylactic agents are provided in, e.g.,Platt, Clin. Lab Med., 7, 289-99 (1987), “Drug Dosage,” J. Kans. Med.Soc., 70(1), 30-32 (1969), and other references described herein (e.g.,Remington's, supra).

[0426] Typically, a nucleic acid composition of the invention comprisesfrom about 1 μg to about 10 mg of at least one nucleic acid of theinvention, including about 1 μg to about 15 mg, including about 1 μg toabout 10 mg, about 500 μg to about 10 mg, about 500 μg to about 5 mg,about 1 mg to about 5 mg, about 2 mg to about 5 mg, about 1 μg to about2 mg, including about 1 μg to about 1 mg, about 1 μg to about 500 μg, 1μg to about 100 μg, 1 μg to about 50 μg, and 1 μg to about 10 μg of thenucleic acid. For delivery of a vector comprising a nucleic acid of theinvention, the same amount(s) can be administered. In one aspect, thecomposition to be administered to a host comprises about 1, 2, 5, or 10mg of a nucleic acid or vector of the invention. A mixture of two ormore nucleic acids of the invention (or mixture of two or more vectors,each encoding a nucleic acid of the invention) can be administered insuch amount(s). The volume of carrier or diluent in which such nucleicacid is administered depends upon the amount of nucleic acid to beadministered. For example, 2 mg nucleic acid is typically administeredin a 1 mL volume of carrier or diluent. The amount of nucleic acid inthe composition depends on the host to which the nucleic acidcomposition is to be administered, the characteristics of the nucleicacid (e.g., gene expression level as determined by the encoded peptide,codon optimization, and/or promoter profile), and the form ofadministration. For example, biolistic or “gene gun” delivery methods ofas little as about 1 μg of nucleic acid dispersed in or on suitableparticles is effective for inducing an immune response even in largemammals such as humans. In some instances, biolistic delivery of atleast about 5 μg, more preferably at least about 10 μg, or more nucleicacid may be desirable. Biolistic delivery of nucleic acids is discussedfurther elsewhere herein.

[0427] For injection of a nucleic acid composition, a larger dose ofnucleic acid typically will be desirable. In general, an injectablenucleic acid composition comprises at least about 1 μg nucleic acid,typically about 5 μg nucleic acid, more typically at least about 25 μgof nucleic acid or at least about 30 μg of nucleic acid, 50 μg ofnucleic acid, usually at least about 75 μg or at least about 80 μg ofthe nucleic acid, preferably at least about 100 μg or at least about 150μg nucleic acid, preferably at least about 500 μg, at least about 1 mg,at least about 2 mg nucleic acid, at least about 5 mg nucleic acid, ormore. In some instances, the injectable nucleic acid composition maycomprise about 0.25-5 mg of the nucleic acid, typically in a volume ofdiluent, carrier, or excipient of about 0.5-1 mL. Commonly, aninjectable nucleic acid solution comprises about 0.5 mg, about 1 mg, 1.5mg, or even about 2 mg nucleic acid, usually in a volume of about 0.25mL, about 0.5 mL, 0.75 mL, or about 1 mL. In one aspect, 2 mg nucleicacid is typically administered in a 1 mL volume of carrier, diluent, orexcipient (e.g., PBS or saline). However, in some instances, lowerinjectable doses (e.g., less than about 5 μg, such as, e.g., about 4 μg,about 3, about 2 μg, or about 1 μg) of the polynucleotide of theinvention are about equally or more effective in producing an antibodyresponse than the above-described higher doses.

[0428] A viral vector composition of the invention can comprise anysuitable number of viral vector particles. The dosage of viral vectorparticles or viral vector particle-encoding nucleic acid depends on thetype of viral vector particle with respect to origin of vector (e.g.,whether the vector is an alphaviral vector, papillomaviral vector, HSVvector, and/or an AAV vector), whether the vector is a transgeneexpressing or recombinant peptide displaying vector, the host, and otherconsiderations discussed above. Generally, with respect to gene transfervectors, the pharmaceutically acceptable composition comprises at leastabout 1×10² viral vector particles in a volume of about 1 mL (e.g., atleast about 1×10² to about 1×10⁸ particles in about 1 mL). Higherdosages also can be suitable (e.g., at least about 1×10⁶, about 1×10⁸,about 1×10⁹, about 1×10¹⁰, or more particles/mL).

[0429] Nucleic acid compositions of the invention can compriseadditional nucleic acids. For example, a nucleic acid can beco-administered with a second immunostimulatory sequence or a secondcytokines/adjuvant-encoding sequence (e.g., a sequence encoding anIFN-gamma and/or a GM-CSF). Examples of such sequences are describedabove.

[0430] The invention further provides a composition comprising aplurality of VLPs (of at least one type) formed from, e.g., recombinantC15/full prM/full E polypeptides or full prM/full E polypeptides of theinvention. Desirably, the composition comprises a dose of VLPssufficient to induce protective immunity in a subject, such as amammalian host. Dosage considerations for VLPs are similar to thosedescribed above with respect to viral vector particles and othercompositions of the invention.

[0431] The invention also provides a composition comprising an aggregateof two or more polypeptides of the invention. Moreover, the inventionprovides a composition comprising a population of one or more multimericpolypeptides of the invention. In particular, recombinant dengueantigens of the invention can form dimers (and in some instancestrimers) in certain conditions and can retain such a multimeric state ina subject, e.g., mammalian host, as shown in the Examples below.

[0432] The invention further provides methods of making and using thepolypeptides, polynucleotides, vectors, and cells of the invention. Inone aspect, the invention provides a method of making a recombinantpolypeptide of the invention by introducing a nucleic acid of theinvention into a population of cells in a culture medium, culturing thecells in the medium (for a time and under conditions suitable fordesired level of gene expression) to produce the polypeptide, andisolating the polypeptide from the cells, culture medium, or both. Thepolypeptide can be isolated from cell lysates, and/or cell culturemedium by first concentrating the culture medium using centrifugalfilters (Amicon), alternatively, by precipitating the polypeptides withammonium sulfate or polyethylene glycol and then resuspending thepolypeptides in PBS or other suitable buffers. The polypeptides can thenbe purified using either size-exclusion chromatography on SephacrylS-400 column (Amersham Biosciences) as described in, e.g., Hjorth, R.and J. Moreno-Lopez. 1982., J. Virol. Methods 5:151-158, or anotheraffinity chromatography, or by centrifugation through 20-60% sucrosegradients as described in, e.g., Konish, E., S. et al., 1992, Virology188:714-720 (see FIGS. 15A-15B). Fractions containing the desiredpolypeptides can be identified by ELISA or SDS-PAGE followed by proteinsilver stain and immunoblotting. The desired fractions are pooled andfurther concentrated. Sucrose in gradient centrifugation fractions canbe removed using PD-10 column (Amersham Biosciences) gel filtration.Additional purification techniques include hydrophobic interactionchromatography (Diogo, M. M, et al., 2001., J Gene Med. 3:577-584) orany other suitable technique known in the art. A variety of polypeptidepurification methods are well known in the art, including those setforth in, e.g., Sandana (1997) BIOSEPARATION OF PROTEINS, AcademicPress, Inc., Bollag et al. (1996) PROTEIN METHODS, 2^(nd) EditionWiley-Liss, NY, Walker (1996) THE PROTEIN PROTOCOLS HANDBOOK HumanaPress, N.J., Harris and Angal (1990) PROTEIN PURIFICATION APPLICATIONS:A PRACTICAL APPROACH IRL Press at Oxford, Oxford, England, Scopes (1993)PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE 3^(rd) Edition SpringerVerlag, NY, Janson and Ryden (1998) PROTEIN PURIFICATION: PRINCIPLES,HIGH RESOLUTION METHODS AND APPLICATIONS, Second Edition Wiley-VCH, NY;and Walker (1998) PROTEIN PROTOCOLS ON CD-ROM Humana Press, NJ. Cellssuitable for polypeptide production are known in the art and arediscussed elsewhere herein (e.g., Vero cells, 293 cells, BHK, CHO, andCOS cells can be suitable). Cells can be lysed by any suitable techniqueincluding, e.g., sonication, microfluidization, physical shear, Frenchpress lysis, or detergent-based lysis. The invention provides a similarmethod of making a polypeptide of the invention comprising inserting avector according to the invention to the cells, culturing the cellsunder appropriate conditions for expression of the nucleic acid from thevector, and isolating the polypeptide from the cells, culture medium, orboth. The cells chosen are based on the desired processing of thepolypeptide and based on the appropriate vector (e.g., E. coli cells canbe preferred for bacterial plasmids, whereas 293 cells can be preferredfor mammalian shuttle plasmids and/or adenoviruses, particularlyE1-deficient adenoviruses).

[0433] In addition to recombinant production, the polypeptides may beproduced by direct peptide synthesis using solid-phase techniques (see,e.g., Stewart et al. (1969) SOLID-PHASE PEPTIDE SYNTHESIS, W H FreemanCo, San Francisco and Merrifield J. (1963) J Am Chem Soc 85:2149-2154).Peptide synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City,Calif.) in accordance with the instructions provided by themanufacturer. For example, subsequences may be chemically synthesizedseparately and combined using chemical methods to produce a polypeptideof the invention or fragments thereof. Alternatively, synthesizedpolypeptides may be ordered from any number of companies that specializein production of polypeptides. Most commonly, polypeptides of theinvention are produced by expressing coding nucleic acids and recoveringpolypeptides, e.g., as described above.

[0434] In another aspect, the invention provides a method of producing apolypeptide of the invention comprising introducing a nucleic acid ofthe invention, a vector of the invention, or a combination thereof, intoa subject, which typically and preferably is a mammal (e.g., a rat, anonhuman primate, a bat, a marmoset, a pig, or a chicken), such that apolypeptide of the invention is expressed in the subject, and thepolypeptide is isolated from the animal or from a byproduct of thesubject. Isolation of the polypeptide from the subject, e.g., animal, oranimal byproduct can be by any suitable technique, depending on thesubject and desired recovery strategy. For example, the polypeptide canbe recovered from sera of mice, monkeys, or pigs expressing thepolypeptide of the invention. Transgenic animals (which preferably aremammals, such as the aforementioned mammals) comprising at least onenucleic acid of the invention also are provided. The transgenic animalcan have the nucleic acid integrated into its host genome (e.g., by anAAV vector, lentiviral vector, biolistic techniques performed withintegration-promoting sequences, etc.) or can have the nucleic acid inmaintained epichromosomally (e.g., in a non-integrating plasmid vectoror by insertion in a non-integrating viral vector). Epichromosomalvectors can be engineered for more transient gene expression thanintegrating vectors. RNA-based vectors offer particular advantages inthis respect.

[0435] The invention additionally provides a method of producing atleast one antibody that binds to at least a portion of a dengue virus.The invention further provides a method of producing at least oneantibody that binds to at least one dengue virus of at least oneserotype, preferably binds to one or more dengue viruses of each of twoor three serotypes, and more preferably binds to one or more dengueviruses of all four virus serotypes, which comprises administering aneffective amount (e.g., antigenic or immunogenic amount) of at least onerecombinant polypeptide of the invention or an antigenic or immunogenicfragment thereof, or an effective amount of a vector or nucleic acidencoding such at least one polypeptide, or composition comprising aneffective amount of such at least one polypeptide or nucleic acid orpolynucleotide encoding said at least polypeptide, to a suitable animalhost or host cell. The host cell is cultured or the animal host ismaintained under conditions permissive for formation of antibody-antigencomplexes. Subsequently produced antibodies are recovered from the cellculture, the animal, or a byproduct of the animal (e.g., sera from amammal). The production of antibodies can be carried out with either atleast one polypeptide of the invention, or a peptide or polypeptidefragment thereof comprising at least about 10 amino acids, preferably atleast about 15 amino acids (e.g., about 20 amino acids), and morepreferably at least about 25 amino acids (e.g., about 30 amino acids) ormore in length. Alternatively, a nucleic acid or vector can be insertedinto appropriate cells, which are cultured for a sufficient time andunder periods suitable for transgene expression, such that a nucleicacid sequence of the invention is expressed therein resulting in theproduction of antibodies that bind to the recombinant antigen encoded bythe nucleic acid sequence. Antibodies thereby obtained can havediagnostic and/or prophylactic uses. The provision of such antibodies,and compositions and pharmaceutical compositions comprising suchantibodies (by use of the principles described above with respect toother compositions and pharmaceutically acceptable compositions) arefeatures of the invention.

[0436] Antibodies produced in response to at least one polypeptide ofthe invention, fragment thereof, or the expression of such at least onepolypeptide by a vector and/or polynucleotide of the invention can beany suitable type of antibody or antibodies. Antibodies provided by theinvention include, e.g., polyclonal antibodies, monoclonal antibodies,chimeric antibodies, humanized antibodies, single chain antibodies, Fabfragments, and fragments produced by a Fab expression library. Methodsof producing polyclonal and monoclonal antibodies are known to those ofskill in the art, and many antibodies are available. See, e.g., CURRENTPROTOCOLS IN IMMUNOLOGY, John Colligan et al., eds., Vols. I-IV (JohnWiley & Sons, Inc., NY, 1991 and 2001 Supplement), and Harlow and Lane(1989) ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Press, NY,Stites et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein,Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.)Academic Press, New York, N.Y., and Kohler and Milstein (1975) Nature256:495-497. Other suitable techniques for antibody preparation includeselection of libraries of recombinant antibodies in phage or similarvectors. See, Huse et al. (1989) Science 246:1275-1281; and Ward et al.(1989) Nature 341:544-546. Specific monoclonal and polyclonal antibodiesand antisera will usually bind with a K_(D) of at least about 0.1 μM,preferably at least about 0.01 μM or better, and preferably, 0.001 μM orbetter.

[0437] Detailed methods for preparation of chimeric (humanized)antibodies can be found in U.S. Pat. No. 5,482,856. Additional detailson humanization and other antibody production and engineering techniquescan be found in Borrebaeck (ed.) (1995) ANTIBODY ENGINEERING, 2^(nd) Ed.Freeman and Co., NY (Borrebaeck); McCafferty et al. (1996) ANTIBODYENGINEERING, A PRACTICAL APPROACH IRL at Oxford Press, Oxford, England(McCafferty), and Paul (1995) ANTIBODY ENGINEERING PROTOCOLS HumanaPress, Towata, N.J. (Paul).

[0438] Humanized antibodies are especially desirable in applicationswhere the antibodies are used as therapeutics and/or prophylactics invivo in mammals (e.g., such as humans) and ex vivo in cells or tissuesthat are delivered to or transplanted into mammals (e.g., humans). Humanantibodies consist of characteristically human immunoglobulin sequences.The human antibodies of this invention can be produced in using a widevariety of methods (see, e.g., Larrick et al., U.S. Pat. No. 5,001,065,and Borrebaeck McCafferty and Paul, supra, for a review). In oneembodiment, the human antibodies of the present invention are producedinitially in trioma cells. Genes encoding the antibodies are then clonedand expressed in other cells, such as nonhuman mammalian cells. Thegeneral approach for producing human antibodies by trioma technology isdescribed by Ostberg et al. (1983), Hybridoma 2:361-367, Ostberg, U.S.Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666. Theantibody-producing cell lines obtained by this method are called triomasbecause they are descended from three cells—two human and one mouse.Triomas have been found to produce antibody more stably than ordinaryhybridomas made from human cells.

[0439] Additional useful techniques for preparing antibodies aredescribed in, e.g., Gavilodono et al., Biotechniques 29(1):128-32,134-6, and 138 (passim) (2000), Nelson et al., Mol. Pathol. 53(3):111-7(2000), Laurino et al. Ann. Clin. Lab. Sci. 29(3):158-66 (1999), Rapley,Mol. Biotechnol. 3(2):139-54 (1995), Zaccolo et al., Int. J. Clin. Lab.Res. 23(4):192-8 (1993), Morrison, Annu. Rev. Immunol. 10:239-65 (1992),“Antibodies, Annigene, and Molecular Mimiery,” Meth. Enzymd. 178 (J. J.Langone, Ed. 1989), Moore, Clin. Chem., 35(9):1849-53 (1989), Rosalki etal., Clin. Chim. Acta 183(1):45-58 (1989), and Tami et al., Am. J. Hosp.Pharm. 43(11):2816-25 (1986), as well as U.S. Pat. Nos. 4,022,878,4,350,683, and 4,022,878. A technique for producing antibodies withremarkably high binding affinities is provided in Border et al., Proc.Natl. Acad. Sci., USA 97(20):10701-05 (2000).

[0440] The invention further provides a method of promoting, inducing,enhancing or modulating, a mammal's immune response to at least onedengue virus of at least one serotype comprising administering animmunogenic amount of at least one polypeptide to a mammal, such ashuman and non-human primates, such that an immune response to the atleast one dengue virus of the at least one serotype in the mammal ispromoted, induced, enhanced, or modulated. Preferably, the polypeptideis administered in a pharmaceutical composition comprising thepolypeptide of the invention and a pharmaceutically acceptable carrieror excipient as described above. Typically, an injectable,pharmaceutical composition comprising a suitable, pharmaceuticallyacceptable carrier (e.g., PBS) and an immunogenic amount of thepolypeptide is delivered intramuscularly, intraperitoneally,subdermally, transdermally, subcutaneously, or intradermally to the hostfor in vivo. Alternatively, biolistic protein delivery techniques(vaccine gun delivery) can be used (examples of which are discussedelsewhere herein). Any other suitable technique also can be used.Polypeptide administration can be facilitated via liposomes (examplesfurther discussed below).

[0441] The invention also provides a method promoting an immune responseto a dengue virus in a subject by administering an antigenic orimmunogenic amount of at least one nucleic acid of the invention and/orat least one nucleic acid vector (NAV) of the invention, preferably in apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an antigenic immunogenic amount of the at least one nucleicacid and/or at least one nucleic acid vector, to the subject.

[0442] While the following discussion is primary directed to nucleicacids, it will be understood that it applies equally (and, indeed,preferably) to nucleic acid vectors of the invention. The nucleic acidcomposition can be administered or delivered to the host by any suitableadministration route. In some aspects of the invention, administrationof the nucleic acid is parenteral (e.g., subcutaneous, intramuscular, orintradermal), topical, or transdermal. The nucleic acid can beintroduced directly into a tissue, such as muscle, by injection using aneedle or other similar device. See, e.g., Nabel et al. (1990), supra);Wolff et al. (1990) Science, 247:1465-1468), Robbins (1996) GENE THERAPYPROTOCOLS, Humana Press, NJ, and Joyner (1993) GENE TARGETING: APRACTICAL APPROACH, IRL Press, Oxford, England, and U.S. Pat. Nos.5,580,859 and 5,589,466. Other methods such as “biolistic” orparticle-mediated transformation (see, e.g., U.S. Pat. No. 4,945,050,U.S. Pat. No. 5,036,006, Sanford et al., J. Particulate Sci. Tech., 5,27-37 (1987), Yang et al., Proc. Natl. Acad. Sci. USA, 87, 9568-72(1990), and Williams et al., Proc. Natl. Acad. Sci. USA, 88, 2726-30(1991)). These methods are useful not only for in vivo introduction ofDNA into a subject, such as a mammal, but also for ex vivo modificationof cells for reintroduction into a subject (which is discussed furtherelsewhere herein).

[0443] For standard gene gun administration, the vector or nucleic acidof interest is precipitated onto the surface of microscopic metal beads.The microprojectiles are accelerated with a shock wave or expandinghelium gas, and penetrate tissues to a depth of several cell layers. Forexample, the AccelTM Gene Delivery Device manufactured by Agacetus, Inc.Middleton Wis. is suitable for use in this embodiment. The nucleic acidor vector can be delivered by such techniques, for example,intramuscularly, intradermally, subdermally, subcutaneously, and/orintraperitoneally. Additional devices and techniques related tobiolistic delivery Int'l Patent Applications WO 99/2796, WO 99/08689, WO99/04009, and WO 98/10750, and U.S. Pat. Nos. 5,525,510, 5,630,796,5,865,796, and 6,010,478,

[0444] The nucleic acid can be delivered in association with atransfection-facilitating agent, examples of which were discussed above.The nucleic acid can be delivered topically and/or by liquid particledelivery (in contrast to solid particle biolistic delivery). Examples ofsuch nucleic acid delivery techniques, compositions, and additionalconstructs that can be suitable as delivery vehicles for the nucleicacids of the invention are provided in, e.g., U.S. Pat. Nos. 5,591,601,5,593,972, 5,679,647, 5,697,901, 5,698,436, 5,739,118, 5,770,580,5,792,751, 5,804,566, 5,811,406, 5,817,637, 5,830,876, 5,830,877,5,846,949, 5,849,719, 5,880,103, 5,922,687, 5,981,505, 6,087,341,6,107,095, 6,110,898, and International Patent Applications WO 98/06863,WO 98/55495, and WO 99/57275, each of which is incorporated herein byreference in its entirety for all purposes.

[0445] The choice of delivery technique and form of the antigen caninfluence the type of immune response observed upon administration. Forexample, gene gun delivery of many antigens is associated with aTh2-biased response (indicated by higher IgG1 antibody titers andcomparatively low IgG2a titers). Advantageously, at least some of theVLPs of the invention are expected to overcome this Th2-bias that can beobserved with the administration of other dengue virus antigens. Thebias of a particular immune response enables the physician or artisan todirect the immune response promoted by administration of the polypeptideand/or polynucleotide of the invention.

[0446] Alternatively, the nucleic acid can be delivered to the host byway of liposome-based gene delivery. Exemplary techniques and principlesrelated to liposome-based gene delivery is provided in, e.g., Debs andZhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques6(7):682-691; Rose U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309;Brigham et al. (1989) Am J Med Sci 298:278-281; Nabel et al. (1990)Science 249:1285-1288; Hazinski et al. (1991) Am J Resp Cell Molec Biol4:206-209; and Wang and Huang (1987) Proc Natl Acad Sci USA84:7851-7855), and Felgner et al. (1987) Proc. Natl Acad. Sci. USA84:7413-7414), each of which is incorporated herein by reference in itsentirety for all purposes. Suitable liposome pharmaceutically acceptablecompositions that can be used to deliver the nucleic acid are furtherdescribed elsewhere herein.

[0447] Any immunogenic amount of nucleic acid can be used. Typically,where the nucleic acid is administered by injection, about 50 micrograms(ug) to 5 mg, usually about 100 ug to about 2.5 mg, typically about 500μg to about 2 mg or about 800 μg to about 1.5 mg, and often about 2 mgor about 1 mg is administered.

[0448] The amount of DNA plasmid for use in these methods whereadministration is via a gene gun, e.g., typically is from about 100 toabout 1000 times less than the amount used for direct injection. Forexample, for gene gun delivery, the amount of DNA plasmid correspondingto the first range above would be from about 50×10⁻⁸ g to 5×10⁻⁵ g (100times less) or from about 50×10⁻⁹ to about 5×10⁻⁶ g. Despite suchsensitivity, preferably at least about 1 μg of the nucleic acid is usedin such biolistic delivery techniques.

[0449] The expression of the nucleic acid sequence encoding therecombinant dengue antigen can be operably linked to any suitablepromoter and/or other expression controls sequences, examples of whichwere described above. For example, expression of the polynucleotideconstruct can be induced by using an inducible on- and off-geneexpression system. Examples of such on- and off-gene expression systemsinclude the Tet-On™ Gene Expression System and Tet-Off™ Gene ExpressionSystem (see, e.g., Clontech Catalog 2000, pg. 110-111 for a detaileddescription of each such system), respectively.

[0450] Delivery of a viral vector of the invention also can promote animmune response to at least one dengue virus of at least one serotype ina subject. Any suitable viral vector, in any suitable concentration, canbe used to induce the immune response. For example, to the subject hostcan be administered a population of retroviral vectors (examples ofwhich are described in, e.g., Buchscher et al. (1992) J. Virol. 66(5)2731-2739, Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992),Sommerfelt et al., (1990) Virol. 176:58-59, Wilson et al. (1989) J.Virol. 63:2374-2378, Miller et al., J. Virol. 65:2220-2224 (1991),Wong-Staal et al, PCT/US94/05700, Rosenburg and Fauci (1993) inFUNDAMENTAL IMMUNOLOGY, THIRD EDITION Paul (ed) Raven Press, Ltd., NewYork and the references therein), an AAV vector (as described in, e.g.,West et al. (1987) Virology 160:38-47, Kotin (1994) Human Gene Therapy5:793-801, Muzyczka (1994) J. Clin. Invst. 94:1351, Tratschin et al.(1985) Mol. Cell. Biol. 5(11):3251-3260, U.S. Pat. Nos. 4,797,368 and5,173,414, and International Patent Application WO 93/24641), or anadenoviral vector (as described in, e.g., Berns et al. (1995) Ann. NYAcad. Sci. 772:95-104; Ali et al. (1994) Gene Ther. 1:367-384; andHaddada et al. (1995) Curr. Top. Microbiol. Immunol. 199 (Pt3):297-306), such that immunogenic levels of expression of the nucleicacid included in the vector thereby occurs in vivo resulting in thedesired immune response. Other suitable types of viral vectors aredescribed elsewhere herein (including alternative examples of suitableretroviral, AAV, and adenoviral vectors).

[0451] Suitable infection conditions for these and other types of viralvector particles are described in, e.g., Bachrach et al., J. Virol.,74(18), 8480-6 (2000), Mackay et al., J. Virol., 19(2), 620-36 (1976),and FIELDS VIROLOGY, supra. Additional techniques useful in theproduction and application of viral vectors are provided in, e.g.,“Practical Molecular Virology: Viral Vectors for Gene Expression” inMETHODS IN MOLECULAR BIOLOGY, vol. 8, Collins, M. Ed., (Humana Press1991), VIRAL VECTORS: BASIC SCIENCE AND GENE THERAPY, 1st Ed.(Cid-Arregui et al., Eds.) (Eaton Publishing 2000), “Viral ExpressionVectors,” in CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, Oldstone etal., Eds. (Springer-Verlag, NY, 1992), and “Viral Vectors” in CURRENTCOMMUNICATIONS IN BIOTECHNOLOGY, Gluzman and Hughes, Eds. (Cold SpringHarbor Laboratory Press, 1988).

[0452] The toxicity and therapeutic efficacy of the vectors that includerecombinant molecules provided by the invention can be determined usingstandard pharmaceutical procedures in cell cultures or experimentalanimals. For example, the artisan can determine the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population) using procedures presented hereinand those otherwise known to those of skill in the art. Nucleic acids,polypeptides, proteins, fusion proteins, transduced cells and otherformulations of the present invention can be administered at a ratedetermined, e.g., by the LD₅₀ of the formulation, and the side-effectsthereof at various concentrations, as applied to the mass and overallhealth of the patient. Administration can be accomplished via single ordivided doses.

[0453] The viral vector can be targeted to particular tissues, cells,and/or organs. Examples of such vectors are described above. E.g., theviral vector or nucleic acid vector can be used to selectively deliverthe nucleic acid sequence of the invention to monocytes, dendriticcells, cells associated with dendritic cells (e.g., keratinocytesassociated with Langerhans cells), T-cells, and/or B-cells. The viralvector can be a replication-deficient viral vector. The viral vectorparticle also can be modified to reduce host immune response to theviral vector, thereby achieving persistent gene expression. Such“stealth” vectors are described in, e.g., Martin, Exp. Mol. Pathol.,66(1):3-7 (1999), Croyle et al., J. Virol., 75(10): 4792-801 (2001),Rollins et al., Hum. Gene Ther., 7(5), 619-26 (1996), Ikeda et al., J.Virol., 74(10):4765-75 (2000), Halbert et al., J. Virol., 74(3), 1524-32(2000), and Int'l Patent Appn WO 98/40509. Alternatively oradditionally, the viral vector particles can be administered by astrategy selected to reduce host immune response to the vectorparticles. Strategies for reducing immune response to the viral vectorparticle upon administration to a host are provided in, e.g., Maione etal., Proc. Natl. Acad. Sci. USA, 98(11), 5986-91 (2001), Morral et al.,Proc. Natl. Acad. Sci. USA, 96(22), 2816-21 (1999), Pastore et al., Hum.Gene Ther., 10(11), 1773-81 (1999), Morsy et al., Proc. Natl. Acad. Sci.USA, 95(14), 7866-71 (1998), Joos et al., Hum. Gene Ther., 7(13),1555-66 (1996), Kass-Eisler et al., Gene Ther., 3(2), 154-62 (1996),U.S. Pat. Nos. 6,093,699, 6,211,160, 6,225,113, U.S. Pat. Appn2001-0066947A1.

[0454] Any suitable population and concentration (dosage) of viralvector particles can be used to induce the immune response in thesubject host. In some aspects of the invention, at least about 1×10²particles are typically used (e.g., the method can comprisesadministering a composition comprising at least from about 1×10²particles/mL to about 1×10⁹ particles/mL of a suitable viral vectorparticle in about 1-2 mL injectable and pharmaceutically acceptablesolution). When delivered to a host, the population of viral vectorparticles is such that the multiplicity of infection (MOI) desirably isat least from about 1 to about 100 and more preferably from at leastabout 5 to about 30. Considerations in viral vector particle dosing aredescribed elsewhere herein.

[0455] The term “prime” generally refers to the administration ordelivery of a polypeptide of the invention (e.g., recombinant denguevirus antigen) or a polynucleotide encoding such polypeptide to a cellculture or population of cells in vitro, or in vivo to a subject or exvivo to tissue or cells of a subject. The first administration ordelivery (primary contact) may not be sufficient to induce or promote ameasurable response (e.g., antibody response), but may be sufficient toinduce a memory response, or an enhanced secondary response. The term“challenge” generally refers to any procedure that induces, promotes, ormodulates an immune response.

[0456] Preferably, the initial delivery or administration of apolypeptide or polynucleotide of the invention to cells or a cellculture in vitro, or in vivo or ex vivo to tissue or cells of a subjectis followed by one or more secondary (usually repeat) administrations ofthe polynucleotide and/or polypeptide. For example, initialadministration of a polypeptide composition can be followed, typicallyat least about 7 days after the initial polypeptide administration (moretypically about 14-35 days or about 2, 4, 6, 12, or 24 months) afterinitial polypeptide administration), with a first repeat administration(“prime boost”) of a substantially similar (if not identical) dose ofthe polypeptide, typically in a similar amount as the firstadministration (e.g., about 5 μg to about 1 mg, or about 5 μg to 0.1 mgof polypeptide in a 1-2 mL injectable and pharmaceutically acceptablesolution). Desirably, a second repeat administration (or “secondaryboost”) is performed with a similar, if not identical, dose of thepolypeptide composition at about 2-9, 3-6 months, 9-18 months, or about12 or 24 months after the initial polypeptide administration.

[0457] Any technique comprising administering a polypeptide of theinvention can also include the co-administration of one or more suitableadjuvants. Examples of suitable adjuvants include Freund's emulsifiedoil adjuvants (complete and incomplete), alum (aluminum hydroxide and/oraluminum phosphate), lipopolysaccharides (e.g., bacterial LPS),liposomes (including dried liposomes and cytokine-containing (e.g.,IFN-γ-containing and/or GM-CSF-containing) liposomes), endotoxins,calcium phosphate and calcium compound microparticles (see, e.g.,International Patent Application WO 00/46147), mycobacterial adjuvants,Arlacel A, mineral oil, emulsified peanut oil adjuvant (adjuvant 65),Bordetella pertussis products/toxins, Cholera toxins, non-ionic blockpolymer surfactants, Corynebacterium granulosum derived P40 component,fatty acids, aliphatic amines, paraffinic and vegetable oils, beryllium,and immunostimulating complexes (ISCOMs—reviewed in, e.g., Höglund etal. “ISCOMs and immunostimulation with viral antigens” in SUBCELLULARBIOCHEMISTRY (Ed. Harris, J. R.) Plenum, N.Y., 1989, pp. 39-68), Moreinet al., “The ISCOM—an approach to subunit vaccines” in RECOMBINANT DNAVACCINES: RATIONALE AND STRATEGY (Ed. Isaacson, R. E.) Marcel Dekker,New York, 1992, pp. 369-386, and Morein et al., Clin Immunotherapeutics3:461-75 (1995)). Recently, monophosphoryl lipid A, ISCOMs with Quil-A,and Syntex adjuvant formulations (SAFs) containing the threonylderivative or muramyl dipeptide also have been under consideration foruse in human vaccines. Numerous types of adjuvants that can be suitablefor co-administration or serial administration with one or morepolypeptides of the invention are known in the art. Examples of suchadjuvants are described in, e.g., Vogel et al., A COMPENDIUM OF VACCINEADJUVANTS AND EXCIPIENTS (2d Ed)(http://www.niaid.nih.gov/aidsvaccine/pdf/compendium.pdf—accessed Jan.24, 2002), Bennet et al., J. Immun Meth 153:31-40 (1992), Bessler etal., Res Immunol, 143(5): 519-25 (1992), Woodard, Lab Animal Sci39(3):222-5 (1989), Vogel, AIDS Res and Human Retroviruses11(10):1277-1278 (1995), Leenaars et al., Vet Immunol Immunopath40:225-241 (1995), Linblak et al., Scandinavian J Lab Animal Sci 14:1-13(1987), Buiting et al. Res Immunol 143(5):541-548 (1992), Gupta andSiber, Vaccine (14):1263-1276 (1996), and U.S. Pat. Nos. 6,340,464,6,328,965, 6,299,884, 6,083,505, 6,080,725, 6,060,068, 5,961,970,5,814,321, 5,747,024, 5,690,942, 5,679,356, 5,650,155, 5,585,099,4,395,394, and 4,370,265.

[0458] Administration of a nucleic acid of the invention also istypically and preferably followed by boosting (at least a prime,preferably at least a prime and secondary boost). A “prime” is typicallythe first immunization. An initial nucleic acid administration can befollowed by a repeat administration of the nucleic acid at least about 7days, more typically and preferably about 14-35 days, or about 2, 4, 6,9, or 12 months, after the initial nucleic acid administration. Theamount administered in the repeat administration is typicallysubstantially similar (if not identical) to the dose of the nucleic acidinitially administered, (e.g., about 50 μg to about 15 or 20 mg, or 1 mgto about 10 mg, or 2-5 mg in a 1-2 mL volume injectable andpharmaceutically acceptable solution).

[0459] Alternatively, the initial administration of the nucleic acid canbe followed by a prime boost of an immunogenic amount of polypeptide atsuch a time. Preferably, in such aspects, a secondary boost also ispreferably performed with nucleic acid and/or polypeptide, in an amountsimilar to that used in the primary boost and/or the initial nucleicacid administration, at about 2-9, 3-6 months, 9-18 months, or about 12or 24 months after the initial polypeptide administration. Any number ofboosting administrations of nucleic acid and/or polypeptide can beperformed.

[0460] The polypeptide, nucleic acid, and/or vector of the invention canbe used to promote any suitable immune response to at least one denguevirus of one or more serotypes in any suitable context. For example, atleast one recombinant polypeptide, nucleic acid, and/or vector can beadministered as a prophylactic in an immunogenic or antigenic amount toa mammal (preferably, a human) that has not been infected with a denguevirus of a particular serotype. Favorably, the administration of the atleast one recombinant polypeptide, nucleic acid, and/or vector induces aprotective immune response against challenge with at least one denguevirus of at least one serotype, and, as such, can be considered a“vaccine” against dengue virus infection by said at least one denguevirus of the at least one serotype. Preferably, the administration ofthe at least one recombinant polypeptide, nucleic acid, and/or vectorinduces a protective immune response against challenge with at least onedengue virus of each at least two serotypes, at least three serotypes,and preferably at least four serotypes and, as such, can be considered a“vaccine” against dengue virus infection by viruses of the at least two,at least three or all four dengue virus serotypes, respectively.

[0461] In an advantage aspect, at least one polypeptide, polynucleotide,and/or vector of the invention is administered to a mammal, preferably ahuman, that has been previously infected with at one dengue virus of atleast one particular serotype, such that a protective immune response(such as, e.g., a neutralizing antibody immune response) to one or moredengue viruses of other serotypes is induced in the mammal (human), mostpreferably without the occurrence of ADE upon administration of the atleast one polynucleotide, polypeptide, and/or vector, as well as uponchallenge with one or more dengue viruses of serotypes other than theserotype of the dengue virus with the mammal was previously infected.The at least one polypeptide, polynucleotide, and/or vector also can beadministered to a mammal (e.g., a human) actively infected with at leastone dengue virus of one serotype, to aid in the production of an immuneresponse against further dengue virus infections. Most preferably, thepolypeptide, nucleic acid, and/or vector is administered in an amountsufficient to induce a protective immune response in a human at risk fordengue virus infection (or at specific risk for DF and/or DHF), or thatis or has been previously infected with a dengue virus of at least oneserotype, to avoid DHF upon secondary infection, preferably without theoccurrence of ADE.

[0462] The polynucleotides and vectors of the invention can be deliveredby ex vivo delivery of cells, tissues, or organs. As such, the inventionprovides a method of promoting an immune response to a dengue viruscomprising inserting at least one nucleic acid of the invention and/or avector of the invention into a population of cells and implanting thecells in a mammal. Ex vivo administration strategies are known in theart (see, e.g., U.S. Pat. No. 5,399,346 and Crystal et al., CancerChemother. Pharmacol., 43(Suppl), S90-S99 (1999)). Cells or tissues canbe injected by a needle or gene gun or implanted into a mammal ex vivo.Briefly, in ex vivo techniques, a culture of cell (e.g., organ cells,cells of the skin, muscle, etc.) or target tissue is provided, orpreferably removed from the host, contacted with the vector orpolynucleotide composition, and then reimplanted into the host (e.g.,using techniques described in or similar to those provided in). Ex vivoadministration of the nucleic acid can be used to avoid undesiredintegration of the nucleic acid and to provide targeted delivery of thenucleic acid or vector. Such techniques can be performed with culturedtissues or synthetically generated tissue. Alternatively, cells can beprovided or removed from the host, contacted (e.g., incubated with) animmunogenic amount of a polypeptide of the invention that is effectivein prophylactically inducing an immune response to a dengue virus(preferably a protective immune response, such as a protectiveneutralizing antibody response) when the cells are implanted orreimplanted to the host. The contacted cells are then delivered orreturned to the subject to the site from which they were obtained or toanother site (e.g., including those defined above) of interest in thesubject to be treated. If desired, the contacted cells may be graftedonto a tissue, organ, or system site (including all described above) ofinterest in the subject using standard and well-known graftingtechniques or, e.g., delivered to the blood or lymph system usingstandard delivery or transfusion techniques. Such techniques can beperformed with any suitable type of cells. For example, in one aspect,activated T cells can be provided by obtaining T cells from a subject(e.g., mammal, such as a human) and administering to the T cells asufficient amount of one or more polypeptides of the invention toactivate effectively the T cells (or administering a sufficient amountof one or more nucleic acids of the invention with a promoter such thatuptake of the nucleic acid into one or more such T cells occurs andsufficient expression of the nucleic acid results to produce an amountof a polypeptide effective to activate said T cells). The activated Tcells are then returned to the subject. T cells can be obtained orisolated from the subject by a variety of methods known in the art,including, e.g., by deriving T cells from peripheral blood of thesubject or obtaining T cells directly from a tumor of the subject. Otherpreferred cells for ex vivo methods include explanted lymphocytes,particularly B cells, antigen presenting cells (APCs), such as dendriticcells, and more particularly Langerhans cells, monocytes, macrophages,bone marrow aspirates, or universal donor stem cells. A preferred aspectof ex vivo administration of a polynucleotide or polynucleotide vectorcan be the assurance that the polynucleotide has not integrated into thegenome of the cells before delivery or readministration of the cells toa host. If desired, cells can be selected for those where uptake of thepolynucleotide or vector, without integration, has occurred, usingstandard techniques known in the art.

[0463] The invention includes a method of inducing an immune response ina subject to at least one dengue virus of at least one serotypecomprising: (a) providing a population of B cells, dendritic cells, orboth; (b) transforming the cells with at least one nucleic acid of theinvention such that the nucleic acid does not integrate into a genome ofany of the cells, and (c) delivering an effective amount of the cellsto-the subject, wherein the cells express the at least one nucleic acidafter delivery and induce an immune response to the at least one denguevirus in the subject. In some such methods, prior to transforming thecells with the nucleic acid, the cells are obtained from a subject, andafter transformation with the at least one nucleic acid, the cells aredelivered to the same subject. Some such methods further comprisedelivering at least one of the following to a subject: 1) polypeptidecomprising GM-CSF or an interferon (IFN), 2) a nucleic acid encodingGM-CSF or an interferon, and 3) a nucleic acid encoding GM-CSF and aninterferon.

[0464] In another aspect, the invention provides a method of inducing animmune response by administering a population of recombinant VLPs orattenuated viruses of the invention, formed by populations ofpolypeptides comprising, e.g., recombinant C15/full prM/full Epolypeptides or full prM/full E polypeptides of the invention. Theadministration of VLPs or attenuated viruses is carried out usingtechniques similar to those used for the administration of polypeptidesand viral vectors, as described above (e.g., VLPs are preferablyadministered in a pharmaceutically acceptable injectable solution intoor through the skin, intramuscularly, or intraperitoneally). The skinand muscle are generally preferred targets for administration of thepolypeptides, vectors, and polynucleotides of the invention, by anysuitable technique. Thus, the delivery of the polypeptide,polynucleotide, or vector of the invention into or through the skin of asubject, e.g., mammal (preferably a human), is a feature of theinvention. Such administration can be accomplished by transdermaldevices, or, more typically, biolistic delivery of the polypeptide,polynucleotide, and/or vector to, into, or through the skin of themammal, or into exposed muscle of the subject. Transdermal devicesprovided by the invention, described elsewhere herein, for example, canbe applied to the skin of a host for a suitable period such thatsufficient transfer of a polynucleotide and/or vector to the mammaloccurs, thereby promoting an immune response to at least one denguevirus. Muscular administration is more typically facilitated byinjection of a liquid solution comprising a polypeptide, polynucleotide,or vector of the invention. Particular cells that can be advantageouslytargeted include dendritic cells, other APCs, B cells, monocytes, Tcells (including T helper cells), and cells associated with such immunesystem cells (e.g., keratinocytes or other skin cells associated withLangerhans cells). Targeting of vectors and nucleic acids of theinvention is described elsewhere herein. Such targeted administrationcan be performed with nucleic acids or vectors comprising nucleic acidsoperably linked to cell and/or tissue-specific promoters, examples ofwhich are known in the art.

[0465] The polynucleotide of the invention can be administered by anysuitable delivery system, such that expression of a recombinant of thepolypeptide occurs in the host resulting in an immune response to adengue virus. For example, an effective amount of a population ofbacterial cells comprising a nucleic acid of the invention can beadministered to a subject, resulting in expression of a recombinantpolypeptide of the invention, and induction of an immune response todengue viruses in the subject, e.g., mammal. Bacterial cells developedfor mammalian gene delivery are known in the art. In another aspect,administration of a polynucleotide or vector (preferably apolynucleotide vector) of the invention is facilitated by application ofelectroporation to an effective number of cells or an effective tissuetarget, such that the nucleic acid and/or vector is taken up by thecells, and expressed therein, resulting in production of a recombinantpolypeptide of the invention therein and subsequent induction of animmune response to dengue viruses in the mammal.

[0466] In some aspects, the nucleic acid, polypeptide, and/or vector ofthe invention is desirably co-administered with an additional nucleicacid or additional nucleic vector comprising an additional nucleic acidthat increases the immune response to a dengue virus upon administrationof the nucleic acid, polypeptide, and/or vector of the invention.Preferably, such a second nucleic acid comprises a sequence encoding aGM-CSF, an interferon (e.g., IFN-gamma), or both, examples of which arediscussed elsewhere herein. Alternatively, the second nucleic acid cancomprise immunostimulatory (CpG) sequences, as described elsewhereherein. GM-CSF, IFN-gamma, or other polypeptide adjuvants also can beco-administered with the polypeptide, polynucleotide, and/or vector.Co-administration in this respect encompasses administration before,simultaneously with, or after, the administration of the polynucleotide,polypeptide, and/or vector of the invention, at any suitable timeresulting in an enhancement of an immune response to a dengue virus.

[0467] Dengue viruses are transmitted through Aedes mosquito bites,posing a significant threat to people living in or visiting tropicalareas. Dengue virus infections are clinically manifested for most of thecases by dengue fever (DF), which is a self-limited fibril illness. Thesevere to fatal dengue hemorrhagic fever/dengue shock syndrome(DHF/DSS), which is associated with a mortality rate between 1-5%, isoften linked to secondary infections.

[0468] Multiple dengue serotypes can be prevalent in one local area, andit is therefore important to test a patient's serum samplessimultaneously against such multiple serotypes, preferably thoseprevalent in the area in which the patient lives. In areas where allfour dengue serotypes may be prevalent, it is important to test apatient's serum samples simultaneously against all 4 serotypes. Fortreatment, it is not always necessary to determine the specific serotypethat has infected a patient; however, it is important to distinguish thespecific infecting virus(es) from other viruses causing hemorrhagicfever. Cultivating or growing DEN viruses in cultures is very tediousand the quality of the virus samples obtained often varies extensively,due to the growth abilities and stability of the different viruses. Thismakes it difficult to produce ELISA plates with consistent antigenquality for multiple (e.g., 2, 3, especially 4) serotypes in each well.Additionally, due to the difficulty of obtaining inactivated viruses,such assay plates are very expensive and for large scale clinicaltesting in poor countries not affordable.

[0469] The invention provides new diagnostic assays using at least onerecombinant chimeric dengue virus antigen polypeptide that displays oneor more conformational epitopes of one or more of the four dengue virusserotypes and/or recognizes one or more antibodies against at least onedengue virus of each of at least one, two, three or four serotypes. Suchrecombinant polypeptides are recognized by type-specific antisera. Suchrecombinant dengue virus antigens of the invention are useful asdiagnostic tools to capture antibodies against one, two, three, andpreferably all 4 dengue serotypes. In a particular aspect, the inventionprovides diagnostic assays using at least one recombinant or syntheticpolypeptide of the invention that displays one or more conformationalepitopes of each of two, three, or four dengue virus serotypes (DEN-1,DEN-2, DEN-3, and/or DEN-4) and/or recognizes one or more antibodiesagainst at least one dengue virus of each of at least, two, three orfour serotypes (e.g., multivalent antigens).

[0470] In a preferred aspect, the invention provides diagnostic assaysusing at least one recombinant or synthetic polypeptide of the inventionthat displays one or more conformational epitopes of each of the four 4DEN virus serotypes and/or recognizes antibodies against at least onedengue virus of each of the four serotypes. As shown below in detail inthe Examples below, such tetravalent antigenic polypeptides induced anantibody response in vivo in subjects and are useful as vaccinecandidates.

[0471] For example, four recombinant PRM15/tE polypeptides (2/7E, 5/21,2G11, and 6E12) were selected to test as diagnostic antigens (seeExample 19 below for details). Alternatively, “full-length” clones(2/7E-D1, 5/217E-D1, 2G11E-D4, and 6E12-D4) can be used. It waspreviously shown for TBE, another flavivirus, that expression of theviral prM and 100% of the E gene can lead to viral-like particle (VLP)formation, physically and antigenically resembling the virus particles.It is believed recombinant C15/full prM/full E (e.g., 2/7E-D1, 5/21-D1,2G11-D4, and 6E12-D4) or full prM/full E clones of the invention formVLPs. Expressed in human 293 cells, VLP-like antigens are secreted intothe medium, which allows for easy antigen isolation.

[0472] The Western blot of FIG. 13A illustrates recognition of the fourtetravalent clones (2/7E, 5/21, 2G11, and 6E12) by type-specificantisera and the expression and secretion of the antigens from human 293cells. For diagnostics, the antigens can be used for a dot blot assay,ELISA, or dipstick EIA. A dot blot assay is an advantageous assay interms of manufacturing, storage and handling. The use of the recombinantmultivalent antigens as a diagnostic tool was demonstrated by a dot blotassay as shown in Example 19. Each of these recombinant tetravalentpolypeptides was well-secreted and recognized by all 4 type-specificanti-DEN antisera and thus can be used to detect serum antibodiesagainst any of the 4 DEN serotypes in a biological human sample obtainedfrom an animal, including a human. Such diagnostic assays advantageouslyallow for the testing of a subject's serum sample simultaneously forantibodies against all four serotypes.

[0473] The invention further provides methods of diagnosing or screeninga composition, preferably a biological sample obtained from a subject(e.g., vertebrate, such as a mammal), such as blood or serum, for thepresence or absence of one or more anti-flavivirus antibodies of one ormore virus serotypes, including one or more antibodies against one ormore flaviviruses or variants thereof that are closely related to one ormore dengue viruses, and especially anti-dengue virus antibodies(including, e.g., antibodies against one or more dengue viruses orvariants thereof). In one such aspect, the invention provides a methodof diagnosing or screening a sample for the presence of one or moretypes of antibodies (or detecting in the composition the presence of oneor more antibodies) that bind to at least one dengue virus of at leastone serotype. The method comprises contacting a sample with apolypeptide of the invention under conditions such that if the samplecomprises antibodies that bind to at least one dengue virus of at leastone serotype at least one anti-dengue virus antibody binds to thepolypeptide to form a mixed composition, contacting the mixedcomposition with at least one affinity-molecule that binds to ananti-dengue virus antibody, removing unbound affinity-molecule from themixed composition, and detecting the presence or absence of affinitymolecules in the composition, wherein the presence of an affinitymolecule is indicative of the presence of antibodies in the sample thatbind to the at least one dengue virus of the at least one serotype.

[0474] In another aspect, the invention provides a method of diagnosing,detecting in, identifying in, selecting from, or screening a sample forthe presence of antibodies that bind or specifically bind to (or reactwith) at least one dengue virus of at least one serotype. In one aspect,such method comprises contacting a sample with a polypeptide of theinvention under conditions such that if the sample comprises antibodiesthat bind to dengue virus at least one anti-dengue virus antibody bindsto the polypeptide to form an antibody-polypeptide complex and detectingthe presence or absence of an antibody-polypeptide complex, wherein thepresence of an antibody-polypeptide complex is indicative of thepresence of antibodies that bind to a dengue virus. In some suchmethods, the method comprises screening, detecting in, selecting from,or diagnosing a sample for the presence of antibodies that specificallybind to or specifically associate with a dengue virus of one or moreserotypes. Preferably, the sample is a biological sample, preferablyobtained from a mammal, which typically is suspected of and/or at riskfor infection with one or more dengue viruses. Any suitable biologicalsample (i.e., that includes a sufficient quantity of antibodies foranalysis, if present) can be used. Typically and preferably, serum froma mammal, typically a human, is obtained and used for such analysis.Alternatively, tissues where antibody concentrations are expected to behigh, such as lymphoid tissues, can be analyzed.

[0475] The invention also includes an immunoassay for at least onedengue virus antibody which comprises the use of a polypeptide of theinvention as a test sample. The above-described methods can further bemodified to form any suitable type of immunoassay, examples of which aredescribed above. Preferred immunoassays in this respect include dot blotassays, ELISA assays (e.g., competitive ELISA assays), and dipstickEIAs. In preparation of such assays, the polypeptide is bound (orassociated with) a solid or semisolid matrix, to promoteantigen-antibody complex formation. The detection of suchantibody-antigen complexes is typically facilitated with a reagentsuitable for visualization, such as dyes used in ELISA and FACS assaysdescribed elsewhere herein. Compositions comprising such elements areprovided by the invention. For example, the invention provides acomposition comprising at least one polypeptide of the invention boundto a solid matrix, and optionally including a reagent for visualizing anantibody bound to the polypeptide.

[0476] The invention also includes a kit for performing such animmunoassay comprising a composition of a polypeptide of the invention,bound to a solid matrix, in combination with a reagent suitable forvisualization of antigen-antibody complexes after incubation of thematrix with a biological sample suspected of comprising anti-denguevirus antibodies.

[0477] A suitable substrate for performing an immunoassay to detect oneor more anti-dengue virus antibodies in a sample composition isadvantageously provided by obtaining cell free medium, aspirated from aculture of cells transformed with a polynucleotide of the invention(including a nucleic acid vector), or infected with a viral vector ofthe invention, which cells at least partially secrete a polypeptide ofthe invention into the cell medium such that the aspirated medium(supernatant) comprises a sufficient amount of polypeptide for use inthe immunoassay. Remarkably, as little as about 10 μl of such a cellsupernatant can be used as a substrate for a sensitive immunoassay,which is able to detect the presence of antibodies to dengue viruses ofmultiple serotypes, and, most preferably, to all four virus serotypes,preferably in a sample of serum obtained from a mammalian host (e.g., ahuman). The inventors contemplate the use of larger amounts of suchsupernatant (e.g., about 20 μl, about 50 μl, about 100 μl, or more), aswell as the use of cell lysates of cells transfected with nucleic acids(or nucleic acid vectors) of the invention, as well as of cells infectedwith viral vectors of the invention. The supernatant can be associatedwith a matrix for performing EIAs (e.g., with an ELISA plate for ELISAassay or with a suitable membrane for dot blot assay) or can be directlyused in an immunoprecipitation or other direct detectionimmunohistochemical technique. Similar techniques that can be modifiedwith reference to the polypeptides of the invention are described in,e.g., U.S. Pat. No. 5,939,254 and other references cited herein.

[0478] The invention also includes a method of identifying the presenceof antibodies to a flavivirus in a biological sample obtained from asubject, such as a mammal, comprising contacting at least onepolypeptide of the invention (or composition comprising at least onesuch polypeptide and a carrier or a solid matrix) with a biologicalsample obtained from the subject under conditions such that an antibodycapable of binding to a flavivirus in the biological sample binds to thepolypeptide and forms an antibody-polypeptide complex; and detecting thepresence of the antibody-polypeptide complex in the biological sample,thereby indicating the presence of antibodies in the biological sample(e.g., blood or serum).

[0479] Pools or libraries of two or more polypeptides of the inventionalso can be used in diagnosis techniques. Alternatively, a polypeptideof the invention can be added to a pool of other molecules (e.g., a poolof polypeptides, such as a collection of viral antigens). Thus, alibrary comprising two or more polypeptides of the invention is afeature of the invention. Another feature of the invention is a libraryof polypeptides of the invention (e.g., a collection of fragments ofpolypeptides of the invention or a collection of substantially identicalpolypeptides of the invention). The polypeptide(s) of the invention canbe used in such libraries for diagnostic techniques (e.g., multiplediagnostic techniques for viral infection and/or other diseasediagnosis). For example, a library of pathogenic antigens from pathogensassociated with fever (or other disease states), comprising at least onepolypeptide of the invention, can be used to diagnose infection of amammal, preferably a human, by reaction of a biological sample obtainedfrom the mammal with such a library in a manner that a detectablebiological reaction between the sample and at least one component of thelibrary will occur, thereby indicating what type of infection the mammalsuffers from. The incorporation of one or more polypeptides of theinvention in diagnostic chips (“protein chips”) for such diagnostictechniques is a feature of the invention.

[0480] In another respect, the invention provides a polypeptide obtainedby recursive sequence recombination (e.g., DNA shuffling and appropriatescreening/selection methods) performed with a nucleic acid sequence ofthe invention (typically with multiple nucleic acid sequences of theinvention and/or multiple wild-type flaviviral, preferably dengue virus,antigen-encoding sequences). For example, the invention provides apolypeptide obtained by a method of recursive sequence recombinationthat comprises recombining at least a first nucleic acid comprising asequence selected from the group of SEQ ID NOS:169-174 and a secondnucleic acid selected from the group of SEQ ID NOS:169-174 and 215-218,to produce a library of recombinant or synthetic nucleic acids, andscreening the resulting library of recombinant or synthetic nucleicacids to identify at least one optimized nucleic acid that encodes arecombinant polypeptide that induces an immune response to at least aportion of dengue viruses of at least one virus serotype in a subjectabout equal to or greater than the immune response induced by apolypeptide encoded by the first nucleic acid, a polypeptide encoded bythe second nucleic acid, or both. Typically, multiple nucleic acidsselected from the first group and from the second group are used togenerate or produce the library of nucleic acids.

[0481] In one embodiment, the invention provides a recombinant orsynthetic polypeptide obtained by a method comprising: (a) recombiningat least a first nucleic acid comprising a polynucleotide sequenceselected from the group of SEQ ID NOS:211-214 and at least a secondnucleic acid, wherein the at least first and second nucleic acids differfrom each other in two or more nucleotides, to produce a library ofrecombinant or synthetic nucleic acids; (b) selecting from or screeningthe library of recombinant or synthetic nucleic acids to identify atleast one recombinant or synthetic nucleic acid that encodes at leastone recombinant or synthetic polypeptide that induces an immune responsein a subject to at least one dengue virus of at least one serotype thatis about equal to or greater than the immune response induced in thesubject against said at least one dengue virus of said at least oneserotype by the polypeptide encoded by the at least first nucleic acidor the at least second nucleic acid or both; and (c) expressing the atleast one recombinant or synthetic nucleic acid to obtain therecombinant or synthetic polypeptide.

[0482] In another embodiment, the invention provides a recombinant orsynthetic polypeptide obtained by a method comprising: (a) recombiningat least a first nucleic acid comprising a polynucleotide sequenceselected from the group of SEQ ID NOS:211-214 and at least a secondnucleic acid, wherein the at least first and second nucleic acids differfrom each other in two or more nucleotides, to produce a library ofrecombinant or synthetic nucleic acids; (b) selecting or screening thelibrary of recombinant or synthetic nucleic acids to identify at leastone recombinant or synthetic nucleic acid that encodes a recombinant orsynthetic polypeptide that induces an immune response in a subject to atleast one dengue virus of at least one serotype that is about equal toor greater than the immune response induced in the subject against saidat least one dengue virus of said at least one serotype by a polypeptideencoded by the at least first nucleic acid and the at least secondnucleic acid; (c) recombining the at least one recombinant or syntheticnucleic acid with at least a third nucleic acid comprising a sequenceselected from the group of SEQ ID NOS:211-214, to produce a secondlibrary of recombinant or synthetic nucleic acid; (d) selecting from orscreening the second library of recombinant or synthetic nucleic acidsto identify at least one further recombinant or synthetic nucleic acidthat encodes at least one further recombinant or synthetic polypeptidethat induces an immune response to at least one dengue of at least oneserotype in a subject that is about equal to or greater than the immuneresponse induced in the subject against said at least one dengue virusof said at least one serotype by the polypeptide encoded by the at leastfirst nucleic acid, the at least second nucleic acid, or the at leastthird nucleic acid, or any combination thereof; and (e) expressing theat least one further recombinant or synthetic to obtain the at least onefurther recombinant or synthetic polypeptide. Some such recombinant orsynthetic polypeptides of the two embodiments above induce an immuneresponse against the polypeptide encoded by the at least first nucleicacid and against the polypeptide encoded by the at least second nucleicacid in a subject. Further, some such recombinant or syntheticpolypeptides of the two embodiments above induce an immune responseagainst at least one dengue virus of each of dengue-1, dengue-2,dengue-3, and dengue-4 virus serotypes in a subject that is about equalto or greater than the immune response induced by the polypeptideencoded by the at least first nucleic acid, by the polypeptide encodedby the at least second nucleic acid, by the polypeptide encoded by theat least third nucleic acid, or by any combination thereof.

[0483] In another embodiment, the invention provides a recombinant orsynthetic polypeptide obtained by a method comprising: (a) recombiningat least a first nucleic acid comprising a polynucleotide sequenceselected from the group of SEQ ID NOS:215-218, and at least a secondnucleic acid, wherein the at least first and second nucleic acids differfrom each other in two or more nucleotides, to produce a library ofrecombinant or synthetic nucleic acids; (b) selecting from or screeningthe library of recombinant or synthetic nucleic acids to identify atleast one recombinant or synthesized nucleic acid that encodes at leastone recombinant or synthetic polypeptide that induces an immune responseto at least one dengue virus of at least one virus serotype in a subjectthat is about equal to or greater than the immune response inducedagainst said at least one dengue virus of said at least one serotype bythe polypeptide encoded by the at least first nucleic acid or the atleast the second nucleic acid or both; and (c) expressing the at leastone recombinant or synthetic nucleic acid to obtain the at leastrecombinant or synthetic polypeptide.

[0484] More generally, the polynucleotides of the invention andfragments thereof can be used as substrates for any of a variety ofrecombination and recursive sequence recombination reactions, inaddition to their use in standard cloning methods as set forth in, e.g.,Ausubel, Berger, and Sambrook, e.g., to produce additionalpolynucleotides or fragments thereof that encode recombinant denguevirus antigens having desired properties. A variety of such reactionsare known, including those developed by the inventors and theirco-workers.

[0485] A variety of diversity generating protocols for generating andidentifying molecules of the invention having one of more of theproperties described herein are available and described in the art.These procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics. While distinctions andclassifications are made in the course of the ensuing discussion forclarity, it will be appreciated that the techniques are often notmutually exclusive. Indeed, the various methods can be used singly or incombination, in parallel or in series, to access diverse sequencevariants.

[0486] The result of any of the diversity-generating proceduresdescribed herein can be the generation of one or more nucleic acids,which can be selected or screened for nucleic acids with or which conferdesirable properties, or that encode proteins with or which conferdesirable properties. Following diversification by one or more of themethods herein, or otherwise available to one of skill, any nucleicacids that are produced can be selected for a desired activity orproperty described herein, including e.g., an ability to induce,promote, enhance, or modulate an immune response against at least onedengue virus of at least one serotype, T cell proliferation and/oractivation, cytokine production (e.g., (e.g., IL-3 production and/orIFN-γ production), the production of antibodies that bind (react) withat least one flavivirus (e.g., dengue virus) of at least one serotype,preferably two, three or four serotypes in a subject, and/or theproduction of neutralizing antibodies against at least one flavivirus(such as a dengue virus) of at least one, and against at least oneflavivirus of each of at least one, two, three or four serotypes in asubject. For example, the desired property may be the ability to induce,promote, modulate or enhance the production of neutralizing antibodiesagainst at least one dengue virus of each of the dengue-1, dengue-2,dengue-3, and dengue-4 serotypes in a subject. This can includeidentifying any activity that can be detected, for example, in anautomated or automatable format, by any of the assays in the art, suchas the assays discussed herein and exemplified in the Examples in theExample section below. A variety of related (or even unrelated)properties can be evaluated, in serial or in parallel, at the discretionof the practitioner.

[0487] Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences that encode polypeptides ofthe invention as described herein are found in the followingpublications and the references cited therein: Soong, N. et al. (2000)“Molecular breeding of viruses” Nat Genet 25(4):436-439; Stemmer, et al.(1999) “Molecular breeding of viruses for targeting and other clinicalproperties” Tumor Targeting 4:1-4; Ness et al. (1999) “DNA Shuffling ofsubgenomic sequences of subtilisin” Nature Biotechnology 17:893-896;Chang et al. (1999) “Evolution of a cytokine using DNA family shuffling”Nature Biotechnology 17:793-797; Minshull and Stemmer (1999) “Proteinevolution by molecular breeding” Current Opinion in Chemical Biology3:284-290; Christians et al. (1999) “Directed evolution of thymidinekinase for AZT phosphorylation using DNA family shuffling” NatureBiotechnology 17:259-264; Crameri et al. (1998) “DNA shuffling of afamily of genes from diverse species accelerates directed evolution”Nature 391:288-291; Crameri et al. (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang et al. (1997) “Directed evolution of an effectivefucosidase from a galactosidase by DNA shuffling and screening” Proc.Natl. Acad. Sci. USA 94:4504-4509; Patten et al. (1997) “Applications ofDNA Shuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Crameri et al. (1996) “Improved green fluorescent protein bymolecular evolution using DNA shuffling” Nature Biotechnology14:315-319; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp.447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al., (1995) “Single-step assembly of a gene and entireplasmid form large numbers of oligodeoxy-ribonucleotides” Gene,164:49-53; Stemmer (1995) “The Evolution of Molecular Computation”Science 270: 1510; Stemmer (1995) “Searching Sequence Space”Bio/Technology 13:549-553; Stemmer (1994) “Rapid evolution of a proteinin vitro by DNA shuffling” Nature 370:389-391; and Stemmer (1994) “DNAshuffling by random fragmentation and reassembly: In vitro recombinationfor molecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

[0488] The term “shuffling” is used herein to indicate recombinationbetween non-identical sequences, in some embodiments shuffling mayinclude crossover via homologous recombination or via non-homologousrecombination, such as via cre/lox and/or flp/frt systems. Shuffling canbe carried out by employing a variety of different formats, includingfor example, in vitro and in vivo shuffling formats, in silico shufflingformats, shuffling formats that utilize either double-stranded orsingle-stranded templates, primer based shuffling formats, nucleic acidfragmentation-based shuffling formats, and oligonucleotide-mediatedshuffling formats, all of which are based on recombination eventsbetween non-identical sequences and are described in more detail orreferenced herein below, as well as other similar recombination-basedformats.

[0489] Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye & Eckstein (1986) “Inhibitionof restriction endonuclease Nci I cleavage by phosphorothioate groupsand its application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

[0490] Additional suitable diversity-generating methods include pointmismatch repair (Kramer et al. (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using Ml 3 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh & Henikoff(1986) “Use of oligonucleotides to generate large deletions” Nucl. AcidsRes. 14: 5115), restriction-selection and restriction-purification(Wells et al. (1986) “Importance of hydrogen-bond formation instabilizing the transition state of subtilisin” Phil. Trans. R. Soc.Lond. A 317: 415-423), mutagenesis by total gene synthesis (Nambiar etal. (1984) “Total synthesis and cloning of a gene coding for theribonuclease S protein” Science 223: 1299-1301; Sakamar and Khorana(1988) “Total synthesis and expression of a gene for the a-subunit ofbovine rod outer segment guanine nucleotide-binding protein(transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al. (1985)“Cassette mutagenesis: an efficient method for generation of multiplemutations at defined sites” Gene 34:315-323; and Grundstrom et al.(1985) “Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis” Nucl. Acids Res. 13: 3305-3316), double-strand breakrepair (Mandecki (1986) “Oligonucleotide-directed double-strand breakrepair in plasmids of Escherichia coli: a method for site-specificmutagenesis” Proc. Natl. Acad. Sci. USA, 83:7177-7181; and Arnold (1993)“Protein engineering for unusual environments” Current Opinion inBiotechnology 4:450-455). Additional details on many of the abovemethods can be found in Methods in Enzymology Volume 154, which alsodescribes useful controls for trouble-shooting problems with variousmutagenesis methods.

[0491] Additional site-mutagenesis techniques are described in, e.g.,Edelman et al., DNA, 2, 183 (1983), Zoller et al., Nucl. Acids Res., 10,6487-5400 (1982), and Veira et al., Meth. Enzymol., 153, 3 (1987)).Other useful mutagenesis techniques include alanine scanning, or randommutagenesis, such as iterated random point mutagenesis induced byerror-prone PCR, chemical mutagen exposure, or polynucleotide expressionin mutator cells (see, e.g., Bomscheueret et al., Biotechnol. Bioeng.,58, 554-59 (1998), Cadwell and Joyce, PCR Methods Appl., 3(6), S136-40(1994), Kunkel et al., Methods Enzymol., 204, 125-39 (1991), Low et al.,J. Mol. Biol., 260, 359-68 (1996), Taguchi et al., Appl. Environ.Microbiol., 64(2), 492-95 (1998), and Zhao et al., Nat. Biotech., 16,258-61 (1998) for discussion of such techniques). Suitable primers forPCR-based site-directed mutagenesis or related techniques can beprepared by the methods described in, e.g., Crea et al., Proc. Natl.Acad. Sci. USA, 75, 5765 (1978).

[0492] Other useful techniques for promoting sequence diversity includePCR mutagenesis techniques (as described in, e.g., Kirsch et al., Nucl.Acids Res., 26(7), 1848-50 (1998), Seraphin et al., Nucl. Acids Res.,24(16), 3276-7 (1996), Caldwell et al., PCR Methods Appl., 2(1), 28-33(1992), Rice et al., Proc. Natl. Acad. Sci. USA. 89(12), 5467-71 (1992)and U.S. Pat. No. 5,512,463), cassette mutagenesis techniques based onthe methods described in Wells et al., Gene, 34, 315 (1985), phagemiddisplay techniques (as described in, e.g., Soumillion et al., Appl.Biochem. Biotechnol., 47, 175-89 (1994), O'Neil et al., Curr. Opin.Struct. Biol, 5(4), 443-49 (1995), Dunn, Curr. Opin. Biotechnol., 7(5),547-53 (1996), and Koivunen et al., J. Nucl. Med., 40(5), 883-88(1999)), reverse translation evolution (as described in, e.g., U.S. Pat.No. 6,194,550), saturation mutagenesis described in, e.g., U.S. Pat. No.6,171,820), PCR-based synthesis shuffling (as described in, e.g., U.S.Pat. No. 5,965,408) and recursive ensemble mutagenesis (REM) (asdescribed in, e.g., Arkin and Yourvan, Proc. Natl. Acad. Sci. USA, 89,7811-15 (1992), and Delgrave et al., Protein Eng., 6(3), 327-331(1993)). Techniques for introducing diversity into a library ofhomologous sequences also are provided in U.S. Pat. Nos. 6,159,687 and6,228,639.

[0493] Further details regarding various diversity generating methodscan be found in the following U.S. patents, PCT publications andapplications, and EPO publications: U.S. Pat. No. 5,605,793 to Stemmer(Feb. 25, 1997), “Methods for In Vitro Recombination;” U.S. Pat. No.5,811,238 to Stemmer et al. (Sep. 22, 1998) “Methods for GeneratingPolynucleotides having Desired Characteristics by Iterative Selectionand Recombination;” U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3,1998), “DNA Mutagenesis by Random Fragmentation and Reassembly;” U.S.Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) “End-ComplementaryPolymerase Reaction;” U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov.17, 1998), “Methods and Compositions for Cellular and MetabolicEngineering;” WO 95/22625, Stemmer and Crameri, “Mutagenesis by RandomFragmentation and Reassembly;” WO 96/33207 by Stemmer and Lipschutz “EndComplementary Polymerase Chain Reaction;” WO 97/20078 by Stemmer andCrameri “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” WO 97/35966by Minshull and Stemmer, “Methods and Compositions for Cellular andMetabolic Engineering;” WO 99/41402 by Punnonen et al. “Targeting ofGenetic Vaccine Vectors;” WO 99/41383 by Punnonen et al. “AntigenLibrary Immunization;” WO 99/41369 by Punnonen et al. “Genetic VaccineVector Engineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination;” WO 00/18906 by Patten etal., “Shuffling of Codon-Altered Genes;” WO 00/04190 by del Cardayre etal. “Evolution of Whole Cells and Organisms by Recursive Recombination;”WO 00/42561 by Crameri et al., “Oligonucleotide Mediated Nucleic AcidRecombination;” WO 00/42559 by Selifonov and Stemmer “Methods ofPopulating Data Structures for Use in Evolutionary Simulations;” WO00/42560 by Selifonov et al., “Methods for Making Character Strings,Polynucleotides & Polypeptides Having Desired Characteristics;”PCT/US00/26708 by Welch et al., “Use of Codon-Varied OligonucleotideSynthesis for Synthetic Shuffling;” and PCT/US01/06775 “Single-StrandedNucleic Acid Template-Mediated Recombination and Nucleic Acid FragmentIsolation” by Affholter.

[0494] Several different general classes of sequence modificationmethods, such as mutation, recombination, etc. are applicable to thepresent invention and set forth, e.g., in the references above andbelow. The following exemplify some of the different types of preferredformats for diversity generation in the context of the presentinvention, including, e.g., certain recombination based diversitygeneration formats.

[0495] Nucleic acids can be recombined in vitro by any of a variety oftechniques discussed in the references above, including e.g., DNAsedigestion of nucleic acids to be recombined followed by ligation and/orPCR reassembly of the nucleic acids. For example, sexual PCR mutagenesiscan be used in which random (or pseudo random, or even non-random)fragmentation of the DNA molecule is followed by recombination, based onsequence similarity, between DNA molecules with different but relatedDNA sequences, in vitro, followed by fixation of the crossover byextension in a polymerase chain reaction. This process and many processvariants is described in several of the references above, e.g., inStemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751.

[0496] Similarly, nucleic acids can be recursively recombined in vivo,e.g., by allowing recombination to occur between nucleic acids in cells.Many such in vivo recombination formats are set forth in the referencesnoted above. Such formats optionally provide direct recombinationbetween nucleic acids of interest, or provide recombination betweenvectors, viruses, plasmids, etc., comprising the nucleic acids ofinterest, as well as other formats. Details regarding such proceduresare found in the references noted above. Whole genome recombinationmethods can also be used in which whole genomes of cells or otherorganisms are recombined, optionally including spiking of the genomicrecombination mixtures with desired library components (e.g., genescorresponding to the pathways of the present invention). These methodshave many applications, including those in which the identity of atarget gene is not known. Details on such methods are found, e.g., in WO98/31837 by del Cardayre et al. “Evolution of Whole Cells and Organismsby Recursive Sequence Recombination;” and in, e.g., PCT/US99/15972 bydel Cardayre et al., also entitled “Evolution of Whole Cells andOrganisms by Recursive Sequence Recombination.”

[0497] Synthetic recombination methods can also be used in whicholigonucleotides corresponding to targets of interest are synthesizedand reassembled in PCR or ligation reactions which includeoligonucleotides which correspond to more than one parental nucleicacid, thereby generating new recombined nucleic acids. Oligonucleotidescan be made by standard nucleotide addition methods, or can be made,e.g., by tri-nucleotide synthetic approaches. Details regarding suchapproaches are found in the references noted above, including, e.g., WO00/42561 by Crameri et al., “Oligonucleotide Mediated Nucleic AcidRecombination;” PCT/US00/26708 by Welch et al., “Use of Codon-VariedOligonucleotide Synthesis for Synthetic Shuffling;” WO 00/42560 bySelifonov et al., “Methods for Making Character Strings, Polynucleotidesand Polypeptides Having Desired Characteristics;” and WO 00/42559 bySelifonov and Stemmer “Methods of Populating Data Structures for Use inEvolutionary Simulations.”

[0498] In silico methods of recombination can be effected in whichgenetic algorithms are used in a computer to recombine sequence stringsthat correspond to homologous (or even non-homologous) nucleic acids.The resulting recombined sequence strings are optionally converted intonucleic acids by synthesis of nucleic acids that correspond to therecombined sequences, e.g., in concert with oligonucleotidesynthesis/gene reassembly techniques. This approach can generate random,partially random or designed variants. Many details regarding in silicorecombination, including the use of genetic algorithms, geneticoperators and the like in computer systems, combined with generation ofcorresponding nucleic acids (and/or proteins), as well as combinationsof designed nucleic acids and/or proteins (e.g., based on cross-oversite selection) as well as designed, pseudo-random or randomrecombination methods are described in WO 00/42560 by Selifonov et al.,“Methods for Making Character Strings, Polynucleotides and PolypeptidesHaving Desired Characteristics” and WO 00/42559 by Selifonov and Stemmer“Methods of Populating Data Structures for Use in EvolutionarySimulations.” Extensive details regarding in silico recombinationmethods are found in these applications. This methodology is generallyapplicable to the nucleic acid sequences and polypeptide sequences ofthe invention.

[0499] Many methods of accessing natural diversity, e.g., byhybridization of diverse nucleic acids or nucleic acid fragments tosingle-stranded templates, followed by polymerization and/or ligation toregenerate full-length sequences, optionally followed by degradation ofthe templates and recovery of the resulting modified nucleic acids canbe similarly used. In one method employing a single-stranded template,the fragment population derived from the genomic library(ies) isannealed with partial, or, often approximately full length ssDNA or RNAcorresponding to the opposite strand. Assembly of complex chimeric genesfrom this population is then mediated by nuclease-base removal ofnon-hybridizing fragment ends, polymerization to fill gaps between suchfragments and subsequent single stranded ligation. The parentalpolynucleotide strand can be removed by digestion (e.g., if RNA oruracil-containing), magnetic separation under denaturing conditions (iflabeled in a manner conducive to such separation) and other availableseparation/purification methods. Alternatively, the parental strand isoptionally co-purified with the chimeric strands and removed duringsubsequent screening and processing steps. Additional details regardingthis approach are found, e.g., in “Single-Stranded Nucleic AcidTemplate-Mediated Recombination and Nucleic Acid Fragment Isolation” byAffholter, PCT/US01/06775.

[0500] In another approach, single-stranded molecules are converted todouble-stranded DNA (dsDNA) and the dsDNA molecules are bound to a solidsupport by ligand-mediated binding. After separation of unbound DNA, theselected DNA molecules are released from the support and introduced intoa suitable host cell to generate a library enriched sequences, whichhybridize to the probe. A library produced in this manner provides adesirable substrate for further diversification using any of theprocedures described herein.

[0501] Any of the preceding general recombination formats can bepracticed in a reiterative fashion (e.g., one or more cycles ofmutation/recombination or other diversity generation methods, optionallyfollowed by one or more selection methods) to generate a more diverseset of recombinant nucleic acids.

[0502] Mutagenesis employing polynucleotide chain termination methodshave also been proposed (see e.g., U.S. Pat. No. 5,965,408, “Method ofDNA reassembly by interrupting synthesis” to Short, and the referencesabove), and can be applied to the present invention. In this approach,double stranded DNAs corresponding to one or more genes sharing regionsof sequence similarity are combined and denatured, in the presence orabsence of primers specific for the gene. The single strandedpolynucleotides are then annealed and incubated in the presence of apolymerase and a chain terminating reagent (e.g., ultraviolet, gamma orX-ray irradiation; ethidium bromide or other intercalators; DNA bindingproteins, such as single strand binding proteins, transcriptionactivating factors, or histones; polycyclic aromatic hydrocarbons;trivalent chromium or a trivalent chromium salt; or abbreviatedpolymerization mediated by rapid thermocycling; and the like), resultingin the production of partial duplex molecules. The partial duplexmolecules, e.g., comprising partially extended chains, are thendenatured and re-annealed in subsequent rounds of replication or partialreplication resulting in polynucleotides which share varying degrees ofsequence similarity and which are diversified with respect to thestarting population of DNA molecules. Optionally, the products, orpartial pools of the products, can be amplified at one or more stages inthe process. Polynucleotides produced by a chain termination method,such as described above, are suitable substrates for any other describedrecombination format.

[0503] Diversity also can be generated in nucleic acids or populationsof nucleic acids using a recombination procedure known as “incrementaltruncation for the creation of hybrid enzymes” (“ITCHY”) described inOstemeier et al. (1999) “A combinatorial approach to hybrid enzymesindependent of DNA homology” Nature Biotech 17:1205. This approach canbe used to generate an initial a library of variants, which canoptionally serve as a substrate for one or more in vitro or in vivorecombination methods. See, also, Ostemeier et al. (1999) “CombinatorialProtein Engineering by Incremental Truncation,” Proc. Natl. Acad. Sci.USA, 96: 3562-67; Ostermeier et al. (1999), “Incremental Truncation as aStrategy in the Engineering of Novel Biocatalysts,” Biological andMedicinal Chemistry, 7: 2139-44.

[0504] Mutational methods which result in the alteration of individualnucleotides or groups of contiguous or non-contiguous nucleotides can befavorably employed to introduce nucleotide diversity. Many mutagenesismethods are found in the above-cited references; additional detailsregarding mutagenesis methods can be found in following, which can alsobe applied to the present invention. For example, error-prone PCR can beused to generate nucleic acid variants. Using this technique, PCR isperformed under conditions where the copying fidelity of the DNApolymerase is low, such that a high rate of point mutations is obtainedalong the entire length of the PCR product. Examples of such techniquesare found in the references above and, e.g., in Leung et al. (1989)Technique 1:11-15 and Caldwell et al. (1992) PCR Methods Applic.2:28-33. Similarly, assembly PCR can be used, in a process whichinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions can occur inparallel in the same reaction mixture, with the products of one reactionpriming the products of another reaction.

[0505] Oligonucleotide directed mutagenesis can be used to introducesite-specific mutations in a nucleic acid sequence of interest. Examplesof such techniques are found in the references above and, e.g., inReidhaar-Olson et al. (1988) Science, 241:53-57. Cassette mutagenesiscan be used in a process that replaces a small region of a doublestranded DNA molecule with a synthetic oligonucleotide cassette thatdiffers from the native sequence. The oligonucleotide can include, e.g.,completely and/or partially randomized native sequence(s).

[0506] Recursive ensemble mutagenesis is a process in which an algorithmfor protein mutagenesis is used to produce diverse populations ofphenotypically related mutants, members of which differ in amino acidsequence. This method uses a feedback mechanism to monitor successiverounds of combinatorial cassette mutagenesis. Examples of this approachare found in Arkin & Youvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815. Exponential ensemble mutagenesis can be used forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants. Small groups of residues in a sequence of interestare randomized in parallel to identify, at each altered position, aminoacids which lead to functional proteins. Examples of such procedures arein Delegrave & Youvan (1993) Biotechnology Research 11:1548-1552.

[0507] In vivo mutagenesis can be used to generate random mutations inany cloned DNA of interest by propagating the DNA, e.g., in a strain ofE. coli that carries mutations in one or more of the DNA repairpathways. These “mutator” strains have a higher random mutation ratethan that of a wild-type parent. Propagating the DNA in one of thesestrains will eventually generate random mutations within the DNA. Suchprocedures are described in the references noted above. Alternatively,in vivo recombination techniques can be used. For example, amultiplicity of monomeric polynucleotides sharing regions of partialsequence similarity can be transformed into a host species andrecombined in vivo by the host cell. Subsequent rounds of cell divisioncan be used to generate libraries, members of which, include a single,homogenous population, or pool of monomeric polynucleotides.Alternatively, the monomeric nucleic acid can be recovered by standardtechniques, e.g., PCR and/or cloning, and recombined in any of therecombination formats, including recursive recombination formats,described above. Other techniques that can be used for in vivorecombination and sequence diversification are described in U.S. Pat.No. 5,756,316.

[0508] Methods for generating multispecies expression libraries havebeen described (in addition to the reference noted above, see, e.g.,Peterson et al. (1998) U.S. Pat. No. 5,783,431 “METHODS FOR GENERATINGAND SCREENING NOVEL METABOLIC PATHWAYS,” and Thompson, et al. (1998)U.S. Pat. No. 5,824,485 METHODS FOR GENERATING AND SCREENING NOVELMETABOLIC PATHWAYS) and their use to identify protein activities ofinterest has been proposed (In addition to the references noted above,see Short (1999) U.S. Pat. No. 5,958,672 “PROTEIN ACTIVITY SCREENING OFCLONES HAVING DNA FROM UNCULTIVATED MICROORGANISMS”). Multispeciesexpression libraries include, in general, libraries comprising cDNA orgenomic sequences from a plurality of species or strains, operablylinked to appropriate regulatory sequences, in an expression cassette.The cDNA and/or genomic sequences are optionally randomly ligated tofurther enhance diversity. The vector can be a shuttle vector suitablefor transformation and expression in more than one species of hostorganism, e.g., bacterial species, eukaryotic cells. In some cases, thelibrary is biased by preselecting sequences which encode a protein ofinterest, or which hybridize to a nucleic acid of interest. Any suchlibraries can be provided as substrates for any of the methods hereindescribed.

[0509] The polynucleotide sequences of the present invention can beengineered by standard techniques to make additional modifications, suchas, the insertion of new restriction sites, the alteration ofglycosylation patterns, the alteration of pegylation patterns,modification of the sequence based on host cell codon preference, theintroduction of recombinase sites, and the introduction of splice sites.

[0510] In some applications, it is desirable to preselect or prescreenlibraries (e.g., an amplified library, a genomic library, a cDNAlibrary, a normalized library, etc.) or other substrate nucleic acidsprior to diversification, e.g., by recombination-based mutagenesisprocedures, or to otherwise bias the substrates towards nucleic acidsthat encode functional products. Libraries can also be biased towardsnucleic acids which have specified characteristics, e.g., hybridizationto a selected nucleic acid probe.

[0511] “Non-Stochastic” methods of generating nucleic acids andpolypeptides are alleged in Short “Non-Stochastic Generation of GeneticVaccines and Enzymes” WO 00/46344. These methods, including proposednon-stochastic polynucleotide reassembly and site-saturation mutagenesismethods are applicable to the present invention as well. Random orsemi-random mutagenesis using doped or degenerate oligonucleotides isalso described in, e.g., Arkin and Youvan (1992) “Optimizing nucleotidemixtures to encode specific subsets of amino acids for semi-randommutagenesis” Biotechnology 10:297-300; Reidhaar-Olson et al. (1991)“Random mutagenesis of protein sequences using oligonucleotidecassettes” Methods Enzymol. 208:564-86; Lim and Sauer (1991) “The roleof internal packing interactions in determining the structure andstability of a protein” J. Mol. Biol. 219:359-76; Breyer and Sauer(1989) “Mutational analysis of the fine specificity of binding ofmonoclonal antibody 51F to lambda repressor” J. Biol. Chem.264:13355-60); and “Walk-Through Mutagenesis” (U.S. Pat. Nos. 5,830,650and 5,798,208, and European Patent 0 527 809).

[0512] It will readily be appreciated that any of the above describedtechniques suitable for enriching a library prior to diversification canalso be used to screen the products, or libraries of products, producedby the diversity generating methods.

[0513] Kits for mutagenesis, library construction and other diversitygeneration methods are also commercially available. For example, kitsare available from, e.g., Stratagene (e.g., QuickChange™ site-directedmutagenesis kit; and Chameleon™ double-stranded, site-directedmutagenesis kit), Bio/Can Scientific, Bio-Rad (e.g., using the Kunkelmethod described above), Boehringer Mannheim Corp., ClonetechLaboratories, DNA Technologies, Epicentre Technologies (e.g., 5 prime 3prime kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), NewEngland Biolabs, Pharmacia Biotech, Promega Corp., QuantumBiotechnologies, Amersham International plc (e.g., using the Ecksteinmethod above), and Anglian Biotechnology Ltd (e.g., using theCarter/Winter method above).

[0514] The above references provide many mutational formats, includingrecombination, recursive recombination, recursive mutation andcombinations or recombination with other forms of mutagenesis, as wellas many modifications of these formats. Regardless of the diversitygeneration format that is used, the nucleic acids of the invention canbe recombined (with each other, or with related (or even unrelated)sequences) to produce a diverse set of recombinant nucleic acids,including, e.g., sets of homologous nucleic acids, as well ascorresponding polypeptides.

[0515] A recombinant nucleic acid produced by recombining one or morepolynucleotide sequences of the invention with one or more additionalnucleic acids using any of the above-described formats alone or incombination forms a part of the invention. The one or more additionalnucleic acids may include another polynucleotide of the invention;optionally, alternatively, or in addition, the one or more additionalnucleic acid can include, e.g., a nucleic acid encoding anaturally-occurring dengue virus prM and/or E protein-encoding sequence,a prM and/or E sequence of another flavivirus, or, e.g., any otherhomologous or non-homologous nucleic acid or fragments thereof (certainrecombination formats noted above, notably those performed syntheticallyor in silico, do not require homology for efficient recombination).

[0516] Desirably, the recombinant polypeptides obtained by theabove-described recombination methods are functional chimericpolypeptides. For example, a polypeptide produced by one of theabove-described methods often and desirably induces an immune responseagainst a polypeptide encoded by the first nucleic acid as well asagainst a polypeptide encoded by the second nucleic acid in a subject.Because multiple, preferably at least three, and more preferably, atleast four, or more, nucleic acids are used in the recursive sequencerecombination techniques of the invention, a polypeptide obtained from anucleic acid product of such recombination reactions typically comprisestwo or more peptide fragments or peptide portions unique to apolypeptide encoded by one of the parental sequences. Each such peptidefragment or peptide portion is an amino acid sequence of one or morecontiguous amino acids, usually at least about 10, at least about 15, atleast about 20, or at least about 30 or more amino acids in length. Suchpeptide fragments or peptide portions can be identified by sequenceanalysis techniques. Examples of peptides of the invention having suchsequence diversity (or complex chimerism) are described in the Examplessection below. The unique peptide fragments or peptide portionscorresponding to any particular peptide encoded by a parental sequenceare separated from each other by peptide fragments or peptide portions,respectively, corresponding to peptides encoded by other parentalnucleic acids. The parental peptide fragments or portions can be anysuitable size. Typically, a parental peptide fragment or portioncomprises at least about 10, at least about 15, at least about 20, andat least about 30 or more amino acids in length. Multiple peptidefragments or peptide portions desirably include epitopes present in thepeptides encoded by the parental nucleic acids. In this respect, apolypeptide expressed from a recombinant nucleotide sequence of theinvention desirably comprises at least about one, two, or more T-cellepitopes and/or antigenic sequences also present in the polypeptideencoded by at least one parental nucleic acid, and, more preferably, byeach parental nucleic acid. Of course, recombination also can result innovel coding sequences not present in any of the parental nucleic acidsequences, and, as such, produce novel epitopes not observed in any ofthe parental nucleic acid sequences.

[0517] Polynucleotides produced by the above-described recombination,mutagenesis, and standard nucleotide synthesis techniques can bescreened for any suitable characteristic, such as the expression of arecombinant polypeptide having any of the desirable characteristicsattendant the novel dengue virus antigens of the invention, which arediscussed in detail elsewhere herein. Polypeptides produced by suchtechniques and having such characteristics are an important feature ofthe invention. For example, the invention provides a recombinantpolypeptide encoded by a recombinant polynucleotide produced byrecursive sequence recombination with any nucleic acid sequence of theinvention that induces an immune response against one or more dengueviruses of one, two, three, or preferably four virus serotypes in asubject. Preferably, the immune response induced by administration orexpression of such a polypeptide in the subject is about equal to orgreater than the immune response induced by a polypeptide encoded by thefirst nucleic acid, the immune response induced by a polypeptide encodedby the second nucleic acid, or both.

[0518] A polypeptide of the invention can comprise any suitable numberof non-dengue virus antigen peptide fragments. Correspondingly, apolynucleotide of the invention can comprise any suitable number ofnucleic acid fragments encoding such non-dengue virus antigen peptidefragments. Such peptide fragments are typically fused to the C or Nterminus of a polypeptide of the invention, as desired, to form a fusionprotein. Such fusion proteins are an important feature of the invention.In general, the invention provides a polypeptide having any of theabove-described characteristics of the invention, that further includesa heterologous fusion partner (non dengue-antigen encoding peptidefragment or peptide portion), that exhibits at least one biologicalproperty that is separately detectable from the rest of the polypeptidein a subject. Numerous types of heterologous fusion partners can beincorporated into the polypeptide in addition to the immunogenic aminoacid sequence (and optional signal sequence) of the invention describedabove.

[0519] A particularly useful fusion partner is a peptide fragment orpeptide portion that facilitates purification of the polypeptide(“polypeptide purification subsequence”). Several types of suitablepolypeptide purification subsequences are known in the art. Examples ofsuch fusion partners include a polyhistidine sequence,histidine-tryptophan modules that allow purification on immobilizedmetals, such as a hexa-histidine peptide, a sequence encoding such a tagis incorporated in the pQE vector available from QIAGEN, Inc.(Chatsworth, Calif.), a sequence which binds glutathione (e.g.,glutathione-S-transferase (GST)), a hemagglutinin (HA) tag(corresponding to an epitope derived from the influenza hemagglutininprotein; Wilson, I. et al. (1984) Cell 37:767), maltose binding proteinsequences, the FLAG epitope utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle, Wash.)—commerciallyavailable FLAG epitopes also are available through Kodak (New Haven,Conn.), thioredoxin (TRX), avidin, and the like. The inclusion of aprotease-cleavable polypeptide linker sequence between the purificationdomain and the immunogenic amino acid sequence or immunogenic amino acidsequence/signal sequence portion of the polypeptide is useful tofacilitate purification of an immunogenic fragment of the fusionprotein. Histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography, as described in Porathet al. (1992) Protein Expression and Purification 3:263-281) while theenterokinase cleavage site provides a method for separating thepolypeptide from the fusion protein. pGEX vectors (Promega; Madison,Wis.) conveniently can be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). Additional examples ofsuch sequences and the use thereof for protein purification aredescribed in, e.g., International Patent Application WO 00/15823. Afterexpression of the polypeptide and isolation thereof by such fusionpartners or otherwise (as described above), protein refolding steps canbe used, as desired, in completing configuration of the maturepolypeptide.

[0520] A fusion protein of the invention also can include one or moreadditional peptide fragments or peptide portions which promote detectionof the fusion protein. For example, a reporter peptide fragment orportion (e.g., green fluorescent protein (GFP), β-galactosidase, or adetectable domain thereof) can be incorporated in the fusion protein.Additional marker molecules that can be conjugated to the polypeptide ofthe invention include radionuclides, enzymes, fluorophores, smallmolecule ligands, and the like.

[0521] A polypeptide of the invention can further be modified by theinclusion of at least one modified amino acid. The inclusion of one ormore modified amino acids may be advantageous in, for example, (a)increasing polypeptide serum half-life, (b) reducing polypeptideantigenicity, or (c) increasing polypeptide storage stability. Aminoacid(s) are modified, for example, co-translationally orpost-translationally during recombinant production (e.g., N-linkedglycosylation at N-X-S/T motifs during expression in mammalian cells) ormodified by synthetic means. Non-limiting examples of a modified aminoacid include a glycosylated amino acid, a sulfated amino acid, aprenlyated (e.g., farnesylated, geranylgeranylated) amino acid, anacetylated amino acid, an acylated amino acid, a PEG-ylated amino acid,a biotinylated amino acid, a carboxylated amino acid, a phosphorylatedamino acid, and the like. References adequate to guide one of skill inthe modification of amino acids are replete throughout the literature.Example protocols are found in Walker (1998) PROTEIN PROTOCOLS ON CD-ROMHumana Press, Towata, N.J. Preferably, the modified amino acid isselected from a glycosylated amino acid, a PEGylated amino acid, afarnesylated amino acid, an acetylated amino acid, a biotinylated aminoacid, an amino acid conjugated to a lipid moiety, and an amino acidconjugated to an organic derivatizing agent.

[0522] Another feature of the invention is a polypeptide comprising animmunogenic amino acid sequence as described above and furthercomprising a targeting sequence other than, or in addition to, a signalsequence. For example, the polypeptide can comprise a sequence thattargets a receptor on a particular cell type (e.g., a monocyte,dendritic cell, or associated cell) to provide targeted delivery of thepolypeptide to such cells and/or related tissues. Signal sequences aredescribed above, and include membrane localization/anchor sequences(e.g., stop transfer sequences, GPI anchor sequences), and the like.

[0523] In another aspect, the polypeptide can comprise a fusion partnerthat promotes stability of the polypeptide, secretion of the polypeptide(other than by signal targeting), or both. For example, the polypeptidecan comprise an immunoglobulin (Ig) domain, such as an IgG polypeptidecomprising an Fc hinge, a CH2 domain, and a CH3 domain, that promotesstability and/or secretion of the polypeptide.

[0524] A fusion protein of the invention can further include additionalimmunogenic amino acid sequences. For example, the fusion protein cancomprise an amino acid sequence that has substantial identity (e.g., atleast about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, 99.5% sequenceidentity) with a sequence fragment or portion of a flavivirus capsidprotein, preferably a dengue virus capsid protein (e.g., DEN-2 orDEN-4), of at least about 20 amino acids in length. For example, thepolypeptide can comprise a fusion protein of an immunogenic amino acidsequence of the invention and a yellow fever virus or adenovirusenvelope, capsid, or other protein. Alternatively or additionally, apolypeptide of the invention (or polynucleotide encoding a polypeptideof the invention) can be administered with one or more dengue capsidproteins, or portions or fragments of such proteins, such as a portionor fragment of a dengue capsid comprising a T cell epitope (examples ofsuch epitopes are known in the art (see, e.g., Gagnon et al. J. Virol,70(1):141-147 (1996)), or one or more polynucleotides encoding suchpolypeptides or peptides. The polypeptide also or alternatively cancomprise, or be administered with one or more dengue virus nonstructuralproteins, or one or more nucleic acids encoding such proteins orsubstantially identical peptides (e.g., having at least about 75%, 80%,85%, 86%, 87%, 88% or 89%, preferably at least about 90%, 91%, 92%, 93%,or 94%, and more preferably at least about 95% (e.g., about 87-95%), 96%97%, 98%, 99%, 99.5% sequence identity). For example, the addition of anon-structural protein (e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and/orNS5 proteins) may increase a T cell response, if it includes one or moreadditional T cell epitopes. The invention includes polypeptides thathave at least about 80%, 85%, 90% or more sequence identity to a WT NSdengue virus protein and uses of such polypeptides as described herein.The co-delivery of polypeptides comprising nonstructural protein B celland/or T cell epitopes (like those found in Ni (also referred to as NS-1or NS1), N3 (NS-3 or NS3), or N5 (NS-5 or NS5)—see, e.g., Mathew et al.J Clin Invest, 98(7):1684-1692 (1996), Okamoto et al. J Gen Virol79:697-704 (1998), Green et al., Virology 234(2):383-386 (1997), andGarcia et al. Am J Trop Med Hyg 56(4):466-70 (1997)), or co-expressionof sequences encoding at least one such polypeptide, can improve thelevel of the multivalent immune response induced by the delivery and/orexpression of the recombinant polypeptides of the invention in subjects,such as mammals.

[0525] The fusion protein peptide fragments or peptide portions can beassociated in any suitable manner. Typically and preferably, the firstand second peptide fragments or portions are covalently associated(e.g., by means of a peptide or disulfide bond). The peptide fragmentsor portions can be directly fused (e.g., the C-terminus of theimmunogenic amino acid sequence can be fused to the N-terminus of apurification sequence or heterologous immunogenic sequence). The fusionprotein can include any suitable number of modified bonds, e.g.,isosteres, within or between the peptide portions. Alternatively, thefusion protein can include a peptide linker between the peptidefragments or portions that includes one or more amino acid sequences notforming part of the biologically active peptide portions. Any suitablepeptide linker can be used. The linker can be any suitable size.Typically, the linker is less than about 30 amino acid residues,preferably less than about 20 amino acid residues, and more preferablyabout 10 or less than 10 amino acid residues. Typically, the linkerpredominantly comprises or consists of neutral amino acid residues.Suitable linkers are generally described in, e.g., U.S. Pat. Nos.5,990,275, 6,010,883, 6,197,946, and European Patent Application 0 035384. If separation of peptide fragments or peptide portions is desirablea linker that facilitates separation can be used. An example of such alinker is described in U.S. Pat. No. 4,719,326. “Flexible” linkers,which are typically composed of combinations of glycine and/or serineresidues, can be advantageous. Examples of such linkers are describedin, e.g., McCafferty et al., Nature, 348, 552-554 (1990), Huston et al.,Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988), Glockshuber et al.,Biochemistry, 29, 1362-1367 (1990), and Cheadle et al., MolecularImmunol., 29, 21-30 (1992), Huston et al., Proc. Natl. Acad. Sci. USA,85, 5879-5883 (1988), Bird et al., Science, 242, 423-26 (1988), and U.S.Pat. Nos. 5,672,683, 6,165,476, and 6,132,992.

[0526] The use of a linker also can reduce undesired immune response tothe fusion protein created by the fusion of the two peptide fragments orpeptide portions, which can result in an unintended MHC I and/or MHC IIepitope being present in the fusion protein. In addition to the use of alinker, identified undesirable epitope sequences or adjacent sequencescan be PEGylated (e.g., by insertion of lysine residues to promote PEGattachment) to shield identified epitopes from exposure. Othertechniques for reducing immunogenicity of the fusion protein of theinvention can be used in association with the administration of thefusion protein include the techniques provided in U.S. Pat. No.6,093,699.

[0527] A recombinant polypeptide of the invention also desirably doesnot comprise irrelevant epitopes (i.e., non-dengue relevant epitopes) orinter-epitope junctions. Such techniques also can be used to preventpresentation of irrelevant epitopes and epitope-junctions. Techniquesfor analyzing epitopes are further provided in the Examples section,which can be used to rationally design recombinant antigens without suchirrelevant and/or undesired epitopes.

[0528] Fragments of polypeptides of the invention also can be useful inpromoting an immune response to a dengue virus in a subject. Theinvention provides such fragments and methods of use thereof. Forexample, the invention provides a fragment of a polypeptide of theinvention that is at least about 75 amino acids in length, and is notidentical to a fragment of a wild-type envelope protein or wild-typeprM/E protein of DEN-1, DEN-2, DEN-3, or DEN-4, wherein the fragmentinduces an immune response to at least one dengue virus of at least oneserotype in a subject. Particular polypeptide fragments induce an immuneresponse to one or more dengue viruses of multiple serotypes (e.g., two,three, or all four known serotypes), including a neutralizing antibodyresponse to one or more dengue viruses of each of two, three or fourserotypes, and most preferably induce a protective immune responseagainst one or more dengue viruses of each of two, three or fourserotypes when administered or expressed appropriately in a subject.

[0529] In addition to encoding and expressing recombinant immunogenicpolypeptides of the invention, the nucleic acids also can be useful forsense and anti-sense suppression of expression (e.g., to controlexpression levels in tissues away from those in which expression of anadministered nucleic acid or vector is desired). A variety of sense andanti-sense technologies are known in the art, see, e.g., Lichtenstein &Nellen (1997) ANTISENSE TECHNOLOGY: A PRACTICAL APPROACH IRL Press atOxford University, Oxford, England, Agrawal (1996) ANTISENSETHERAPEUTICS Humana Press, NJ, and references cited therein.

[0530] The invention further provides nucleic acids that comprise anucleic acid sequence that is the substantial complement (i.e.,comprises a sequence that complements at least about 90%, preferably atleast about 95%), and more preferably the complement, of any of theabove-described nucleic acid sequences. Such complementary nucleic acidsequences are useful in probes, production of the nucleic acid sequencesof the invention, and as antisense nucleic acids for hybridizing tonucleic acids of the invention. Short oligonucleotide sequencescomprising sequences that complement the nucleic acid, e.g., of about15, 20, 30, or 50 bases (preferably at least about 12 bases), whichhybridize under highly stringent conditions to a nucleic acid of theinvention are useful as probes (e.g., to determine the presence of anucleic acid of the invention in a particular cell or tissue and/or tofacilitate the purification of nucleic acids of the invention).Polynucleotides comprising complementary sequences also can be used asprimers for amplification of the nucleic acids of the invention.

[0531] The invention further provides a fragment of a nucleic acid ofthe invention that comprises a sequence that encodes a uniquesubsequence in a polypeptide selected from the group of SEQ ID NOS:1-49and 153-155 as compared to any of SEQ ID NOS:338-341. Also provided is afragment of a nucleic acid of the invention that comprises a sequencethat encodes a unique subsequence in a polypeptide selected from thegroup of SEQ ID NOS:65-116 as compared to any of SEQ ID NOS:149-152.Also provided is a fragment of a nucleic acid of the invention thatcomprises a sequence that encodes a unique subsequence in a polypeptideselected from the group of SEQ ID NOS:139-148, 236-253, 343, and 345 ascompared to any of SEQ ID NOS:227-230.

[0532] The invention also provides a fragment of a nucleic acid of theinvention that comprises a unique sequence of nucleotides of at leastabout 300, preferably at least about 400, more preferably at least about600, desirably at least about 900, and more desirably at least about1200 nucleotides from a sequence selected from any of SEQ IDNOS:285-330, as compared to a wild-type dengue tE-polypeptide encodingsequence selected from any of SEQ ID NOS:338-341 and similar knowndengue virus truncated E polypeptide-encoding nucleotide sequences,including those available in GenBank, and more preferably as compared toany known wild-type flaviviral truncated E polypeptide-encodingnucleotide sequence. Such fragment induces an immune response to atleast one dengue virus of at least one serotype in a subject whenadministered and is useful in, among other things, methods of inducingan immune response in the subject and cells thereof against such atleast one dengue virus (e.g., neutralizing Ab response against at leastone dengue virus serotype).

[0533] In another aspect, the invention provides a fragment of a nucleicacid of the invention that comprises a unique sequence of nucleotides ofat least about 300, preferably at least about 400, more preferably atleast about 600, desirably at least about 900, and more desirably atleast about 1200 nucleotides from a sequence selected from any of SEQ IDNOS:156-200 and 235, as compared to a wild-type denguePRM15/tE-polypeptide encoding sequence selected from any of SEQ IDNOS:149-152 and similar known dengue virus PRM15/tE polypeptide-encodingnucleotide sequences, including those available in GenBank, and morepreferably as compared to any known wild-type flaviviral PRM15/tEpolypeptide-encoding nucleotide sequence. Such fragment induces animmune response in a subject to at least one dengue virus of at leastone serotype and is useful in, among other things, methods of inducingan immune response in a subject or population of the subject's cellsagainst at least one dengue virus of at least one serotype (e.g.,neutralizing Ab response against one or more viruses of one or moredengue virus serotypes).

[0534] In another aspect, the invention provides a fragment of a nucleicacid of the invention that comprises a unique sequence of nucleotides ofat least about 300, preferably at least about 400, more preferably atleast about 600, desirably at least about 900, and more desirably atleast about 1200 nucleotides from a sequence selected from any of SEQ IDNOS:201-210, 211-218, 254-271, 342, and 344, as compared to a non-codonoptimized WT C15/full prM/full E-polypeptide encoding sequence selectedfrom any of SEQ ID NOS:227-230 and a known dengue virus C15/fullprM/full E polypeptide-encoding nucleotide sequences, including thoseavailable in GenBank, and more preferably as compared to any known WTflaviviral C15/full prM/full E polypeptide-encoding nucleotide sequence.Such fragment induces an immune response in a subject or population ofits cells to at least one dengue virus of at least one serotype and isuseful in, among other things, methods of inducing an immune response insuch subject or cells against at least one dengue virus of at least oneserotype (e.g., neutralizing Ab response against one or more serotypes).

[0535] The invention also provides a nucleic acid that selectivelyhybridizes to at least one of SEQ ID NOS:285-330, or the complementthereof, than as compared to a wild-type dengue tE-polypeptide encodingsequence selected from any of SEQ ID NOS:338-341 and a known denguevirus truncated E polypeptide-encoding nucleotide sequences, includingthose in GenBank, and more preferably as compared to any known wild-typeflaviviral truncated E polypeptide-encoding nucleotide sequence, or thecomplement thereof, as applicable.

[0536] The invention also provides a nucleic acid that selectivelyhybridizes to at least one of SEQ ID NOS:156-200, 211-214, and 235, orthe complement thereof, as compared to a wild-type denguePRM15/tE-polypeptide encoding sequence selected from any of SEQ IDNOS:149-152 and similar known dengue virus PRM15/tE polypeptide-encodingnucleotide sequences, including those available in GenBank, and morepreferably as compared to any known wild-type flaviviral PRM15/tEpolypeptide-encoding nucleotide sequence, or the complement thereof, asapplicable.

[0537] The invention also provides a nucleic acid that selectivelyhybridizes to at least one of SEQ ID NOS:201-210, 215-218, 254-271, 342,and 344, or the complement thereof, as compared to a non-codon optimizedwild-type dengue C15/full prM/full E-polypeptide encoding sequenceselected from any of SEQ ID NOS:227-230 and a known dengue virusC15/full prM/full E polypeptide-encoding nucleotide sequences, includingthose available in GenBank, and more preferably as compared to any knownwild-type flaviviral C15/full prM/full E polypeptide-encoding nucleotidesequence, as applicable.

[0538] The invention further provides a composition and/or a nucleicacid obtained by cleaving a nucleic acid of the invention. The nucleicacid can be cleaved by mechanical, chemical, or enzymatic cleavage.Techniques for cleavage of nucleic acids are known in the art. Cleavageby enzymatic cleavage, particularly endonuclease, exonuclease digestion,RNAse digestion, or DNAse (e.g., benzon nuclease, such as Benzonase®)digestion of the nucleic acid. The composition also can comprise theproducts of cleaving multiple nucleic acids of the invention by suchtechniques.

[0539] The invention further provides a composition and/or nucleic acidproduced by a process that comprises incubating a nucleic acid of theinvention in the presence of nucleotide triphosphates (NTPs, preferablydNTPs) and a nucleic acid polymerase. Typically, and preferably, thepolymerase is a thermostable polymerase, such as a Taq polymerase.

[0540] In another aspect, the invention provides a library or pool ofnon-identical nucleic acids of the invention and/or a library of nucleicacids comprising at least one nucleic acid of the invention. Forexample, the invention provides a library of nucleic acids comprising atleast one nucleic acid having substantial identity (e.g., at least about75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at least about 90%, 91%,92%, 93%, or 94%, and more preferably at least about 95% (e.g., about87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity) with at least onepolynucleotide sequence selected from the group of SEQ ID NOS:156-218,235, 254-271, 285-330, 342, and 344.

[0541] In another context, the invention provides a library ofnon-identical nucleic acids that have substantial identity (e.g., atleast about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequence identity) withat least one sequence selected from SEQ ID NOS:156-218, 235, 254-271,285-330, 342, and 344,. For example, the library in either case might bea library obtained by the above-described recursive sequencerecombination techniques.

[0542] Thus, for example, the invention provides a compositioncomprising a library of nucleic acids obtained by a method comprisingrecombining at least a first nucleic acid comprising a sequence selectedfrom SEQ ID NOS:211-214 and/or 215-218, and at least a second nucleicacid, wherein the first and second nucleic acids differ from each otherin two or more nucleotides, to produce a library of recombinant orsynthetic nucleic acids. The invention also provides nucleic acidsproduced by similar recombination reactions using any nucleic acid ofthe invention.

[0543] The invention also provides a method of recombination comprisingsubjecting at least one nucleic acid sequence of the invention torecursive sequence recombination with at least one additional nucleicacid, as described above. Regardless of how the library or pool isproduced, the library or pool can comprises any suitable number ofnucleic acid species therein. For example, the library can comprise atleast about 2, 5, 10, 50 or more non-identical nucleic acids of theinvention. The library can be inserted into one or more cells, e.g., bylibrary transfection techniques. Such a population of cells is alsocontemplated.

[0544] The invention also provides a composition comprising such alibrary. For example, a library of nucleic acids as described above canbe used for diagnosis of gene expression (e.g., by way of “gene chip”technology well-known in the art). Such libraries can be subject toexpression, hybridization, or any other form of analysis for anysuitable diagnostic purpose.

[0545] The invention also provides a method of producing a modifiednucleic acid comprising mutating a nucleic acid of the invention (e.g.,a nucleic acid selected from the group of SEQ ID NOS:156-218, 235,254-271, 285-330, 342, and 344,). The invention further provides amodified nucleic acid produced by this method. Methods for mutatingnucleic acids of the invention are described elsewhere herein.

[0546] The polynucleotide of the invention can be in the form of anaptamer (as described in, e.g., Famulok andMayer—http://www.chemie.uni-bonn.de/oc/ak_fa/publications/CTMI-paper.pdf),capable of binding to suitable targets. The nucleic acids of theinvention also can be used to form triplex-forming inhibitorynucleotides. The nucleic acid also can be conjugated to a DNA bindingdomain, such that they silence gene expression of undesired genes (e.g.,act as gene decoys).

[0547] The invention also provides protein mimetics of the polypeptidesof the invention. Peptide mimetics are described in, e.g., U.S. Pat. No.5,668,110 and the references cited therein. Furthermore, the fusionprotein can be modified by the addition of protecting groups to the sidechains of one or more the amino acids of the fusion protein. Suchprotecting groups can facilitate transport of the fusion peptide throughmembranes, if desired, or through certain tissues, for example, byreducing the hydrophilicity and increasing the lipophilicity of thepeptide. Examples of suitable protecting groups include ester protectinggroups, amine protecting groups, acyl protecting groups, and carboxylicacid protecting groups, which are known in the art (see, e.g., U.S. Pat.No. 6,121,236). Synthetic fusion proteins of the invention can take anysuitable form. For example, the fusion protein can be structurallymodified from its naturally occurring configuration to form a cyclicpeptide or other structurally modified peptide. The polypeptide of theinvention also can be linked to one or more nonproteinaceous polymers,typically a hydrophilic synthetic polymer, e.g., polyethylene glycol(PEG), polypropylene glycol, or polyoxyalkylene, as described in, e.g.,U.S. Pat. Nos. 4,179,337, 4,301,144, 4,496,689, 4,640,835, 4,670,417,and 4,791,192, or a similar polymer such as polyvinylalcohol orpolyvinylpyrrolidone (PVP). As discussed above, the polypeptide can besubject to common protein modifications, such as carboxylation,glycosylation, hydroxylation, lipid or lipid derivative-attachment,methylation, myristylation, phosphorylation, and sulfation. Otherpost-translational modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formylation, GPI anchor formation, iodination, oxidation, proteolyticprocessing, prenylation, racemization, selenoylation, arginylation, andubiquitination. Other common protein modifications are described in,e.g., Creighton, supra, Seifter et al. (1990) Meth Enzymol 18:626-646,and Rattan et al. (1992) Ann NY Acad Sci 663:48-62. Such modificationsare usually the result of post-translational modifications that occur inrecombinant polypeptides of the invention (alternatively, suchmodifications can be carried out synthetically). Post-translationalmodifications for polypeptides expressed from nucleic acids in hostcells vary depending what kind of host or host cell type the peptide isexpressed in. For instance, glycosylation often does not occur inbacterial hosts such as E. coli and varies considerably in baculovirussystems as compared to mammalian cell systems. Accordingly, whenglycosylation is desired, a polypeptide should be expressed (produced)in a glycosylating host, generally a eukaryotic cell (e.g., a mammaliancell or an insect cell). Additional and particularly preferred proteinmodifications are discussed elsewhere herein. Modifications to thepolypeptide can be verified by any suitable technique, including, e.g.,x-ray diffraction, NMR imaging, mass spectrometry, and/or chromatography(e.g., reverse phase chromatography, affinity chromatography, or GLC).

[0548] The polypeptide also or alternatively can comprise any suitablenumber of non-naturally occurring amino acids (e.g., β amino acids)and/or alternative amino acids (e.g., selenocysteine), or amino acidanalogs, such as those listed in the Manual of Patent ExaminingProcedure §2422 (7th Revision—2000), which can be incorporated byprotein synthesis, such as through solid phase protein synthesis (asdescribed in, e.g., Merrifield (1969) Adv Enzymol 32:221-296 and otherreferences cited herein).

[0549] Recently, the production of fusion proteins comprising aprion-determining domain has been used to produce a protein vectorcapable of non-Mendelian transmission to progeny cells (see, e.g., Li etal., J. Mol. Biol., 301(3), 567-73 (2000)). The inclusion of suchprion-determining sequences in a fusion protein comprising immunogenicamino acid sequences of the invention is contemplated, ideally toprovide a hereditable protein vector comprising the fusion protein thatdoes not require a change in the host's genome.

[0550] The invention also provides a polypeptide which comprises anamino acid sequence of at least about 45 amino acids in length,preferably at least about 55 amino acids in length, and more preferablyat least about 80 amino acids in length, corresponding to a fragment ofa polypeptide of any one of SEQ ID NOS:139-148, 236-253, 343, and 345,wherein the amino acid sequence is unique as compared to a polypeptideencoded by any of SEQ ID NOS:215-217, and known dengue virus C15/fullprM/full E polypeptides. In addition, the invention provides apolypeptide which comprises an amino acid sequence of at least about 45amino acids in length, preferably at least about 55 amino acids inlength, and more preferably at least about 80 amino acids in length,corresponding to a fragment of a polypeptide of any one of SEQ IDNOS:156-200 and 235, wherein the amino acid sequence is unique ascompared to a polypeptide encoded by any of SEQ ID NOS:211-214, andknown dengue virus PRM15/tE polypeptides.

[0551] In another aspect, the invention provides a polypeptide which isspecifically bound by polyclonal antisera raised against at least oneantigen, the at least one antigen comprising an amino acid sequenceselected from the group of SEQ ID NOS:1-49 and 153-155, or an antigenicor immunogenic fragment thereof, wherein said antigenic or immunogenicpolypeptide fragment induces an immune response in a subject against atleast one dengue virus of at least one virus serotype that is aboutequal to or greater than the immune response induced in the mammaliancell by a antigenic or immunogenic polypeptide fragment of the at leastone dengue virus of the at least one serotype; wherein the polyclonalantisera is subtracted with at least one of (1) a truncated envelopeprotein selected from the group of SEQ ID NOS:338-341 and (2) atruncated envelope protein comprising an amino acid sequence fragment ofa known dengue virus truncated E polypeptide, wherein said amino acidsequence fragment has a length substantially identical (e.g., having atleast about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96% 97%, 98%, 99%, 99.5% sequence identity) to the truncated Eprotein of any of SEQ ID NOS:338-341.

[0552] The invention also includes an antibody or antisera thatspecifically binds a polypeptide, the polypeptide comprising an aminoacid sequence selected from the group of SEQ ID NOS:1-49 and 153-155,wherein the antibody or antisera does not specifically bind to apolypeptide comprising one or more of: the polypeptides of SEQ IDNOS:338-341 and known dengue virus truncated E proteins.

[0553] Further provided is a recombinant or synthetic polypeptide whichis specifically bound by a polyclonal antisera raised against at leastone antigen, the antigen comprising an amino acid sequence selected fromthe group of SEQ ID NOS:65-116, or a fragment thereof, wherein theantisera is subtracted with polypeptides encoded by SEQ ID NOS:149-152,and known dengue virus PRM15/tE polypeptides.

[0554] Further provided is a recombinant or synthetic polypeptide whichis specifically bound by a polyclonal antisera raised against at leastone antigen, the antigen comprising an amino acid sequence selected fromthe group of SEQ ID NOS:139-148, 236-253, 343, and 345, or a fragmentthereof, wherein the antisera is subtracted with polypeptides encoded bySEQ ID NOS:227-230, and known dengue virus C15/full prM/full Epolypeptides.

[0555] In general, the polypeptides of the invention provide structuralfeatures that can be recognized, e.g., in immunological assays. Theproduction of antisera comprising at least one antibody (for at leastone antigen) that specifically binds a polypeptide of the invention, andthe polypeptides which are bound by such antisera, are features of theinvention. Binding agents, including antibodies described herein, maybind the dengue antigen polypeptides of the invention and/or fragmentsthereof with affinities of about 1×10² M⁻¹ to about 1×10¹⁰ M⁻¹ (i.e.,about 10⁻²-10⁻¹⁰ M) or greater, including about 10⁴ to 10⁶ M⁻¹, about10⁶ to 10⁷ M⁻¹, or about 10⁸ M⁻¹ to 10⁹ M⁻¹ or 10¹⁰ M⁻¹. Conventionalhybridoma technology can be used to produce antibodies having affinitiesof up to about 10⁹ M⁻¹. However, new technologies, including phagedisplay and transgenic mice, can be used to achieve higher affinities(e.g., up to at least about 10¹² M⁻¹). In general, a higher bindingaffinity is advantageous.

[0556] In order to produce antiserum or antisera for use in animmunoassay, at least one immunogenic polypeptide (orpolypeptide-encoding polynucleotide) of the invention is produced andpurified as described herein. For example, recombinant polypeptide maybe produced in a mammalian cell line. Alternatively, an inbred strain ofmice can immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant or alum, and a standardmouse immunization protocol (see Harlow and Lane, supra, for a standarddescription of antibody generation, immunoassay formats and conditionsthat can be used to determine specific immunoreactivity). Alternatively,at least one synthetic or recombinant polypeptide derived from at leastone polypeptide sequence disclosed herein or expressed from at least onepolynucleotide sequence disclosed herein can be conjugated to a carrierprotein and used as an immunogen for the production of antiserum.Polyclonal antisera typically are collected and titered against theimmunogenic polypeptide in an immunoassay, for example, a solid phaseimmunoassay with one or more of the immunogenic proteins immobilized ona solid support. In the above-described methods where novel antibodiesand antisera are provided, antisera resulting from the administration ofthe polypeptide (or polynucleotide and/or vector) with a titer of about10⁶ or more typically are selected, pooled and subtracted with thecontrol co-stimulatory polypeptides to produce subtracted pooled titeredpolyclonal antisera.

[0557] Cross-reactivity of antibodies can be determined using standardtechniques, such as competitive binding immunoassays and/or parallelbinding assays, and standard calculations for determining the percentcross-reactivity. Usually, where the percent cross-reactivity is atleast 5-10× as high for the test polypeptides, the test polypeptides aresaid to specifically bind the pooled subtracted antisera or antibody.

[0558] Antisera raised or induced by an immunizing antigen may bindrelated antigens (e.g., cross-react). In such instance, thecross-reacting antigens comprise the same or substantially equivalentepitopes or comprise epitopes that are, e.g., sufficiently similar inshape to bind the same antibody.

[0559] The invention also provides polynucleotide consensus sequencesderived from a comparison of two or more of the polynucleotide sequencesdescribed herein (e.g., a consensus sequence obtained by comparison oftwo or more sequences selected from, e.g., SEQ ID NOS:156-218, 235,254-271, 285-330, 342, and 344). Preferably, the nucleic acid provides anon-naturally-occurring or recombinant polynucleotide comprising asequence obtained by selection of nucleic acids from such a consensussequence. The invention also provides a polypeptide consensus sequenceobtained by similar analysis of at least two polypeptides of theinvention (e.g., by comparison of any two amino acids selected from thegroup of SEQ ID NOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and345). The invention further provides a polypeptide comprising a sequenceaccording to the sequence pattern (formula) obtained by such consensussequence analysis. Examples of such formulas are described herein, suchas the one obtained by standard alignment of the polypeptides.

[0560] The invention also provides polypeptides and/or polynucleotidesthat comprise a sequence that has substantial local sequence identity,in contrast to global/overall sequence identity (discussed primarilyherein), to one of the sequences specifically disclosed herein. Localsequence identity can be determined using local sequence alignmentsoftware, e.g., the BLAST programs described above, the LFASTA program,or, more preferably, the LALIGN program. Preferably, the LALIGN programusing a BLOSUM50 matrix analysis is used for amino acid sequenceanalysis, and a +5 match/−4 mismatch analysis is used for polynucleotidesequence analysis. Gap extension and opening penalties are preferablythe same as those described above with respect to analysis with theALIGN program. For LALIGN (or other program) analysis using k-tup valuesettings (also referred to as “k-tuple” or ktup values), a k-tup valueof 0-3 for proteins, and 0-10 (e.g., about 6) for nucleotide sequences,is preferred. The invention provides in this respect, for example, apolynucleotide that comprises a sequence that has substantial localidentity (i.e., a percent identity similar to the percent identitiesdiscussed with respect to substantially identical sequences) (e.g.,having at least about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably atleast about 90%, 91%, 92%, 93%, or 94%, and more preferably at leastabout 95% (e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% sequenceidentity) over a sequence of at least about 300, desirably at leastabout 450, more desirably at least about 600, preferably at least about900, and more preferably at least about 1200 nucleotides, with animmunogenic amino acid-encoding portion of a nucleic acid of theinvention, despite lacking substantial identity due to gap penaltiesand/or differences in length of the analyzed sequences. Similarly, theinvention provides a polypeptide that comprises an amino acid sequenceof at least about 10, preferably at least about 20, more preferably atleast about 50, favorably at least about 100, more favorably at leastabout 200, or more (e.g., at least about 300, 350, or 375) amino acidresidues that has substantial local identity with an immunogenic aminoacid sequence of one of the recombinant polypeptides of the invention(e.g., a recombinant tE sequence, full E sequence, PRM15/tE sequence,C15/full prM/tE sequence, C15/full prM/full E sequence, etc.).

[0561] A recombinant polypeptide of the invention desirably, though notnecessarily, exhibits primary, secondary, and/or tertiary structuralsimilarity to a wild-type dengue virus polypeptide, or at least afragment thereof of similar length to the recombinant polypeptide. Forexample, a recombinant tE, full E, PRM15/tE, C15/full prM/tE, orC15/full prM/full E polypeptide may exhibit primary, secondary, and/ortertiary structural similarity to a wild-type dengue virus polypeptidetE, full E, PRM15/tE, C15/full prM/tE, or C15/full prM/full Epolypeptide, respectively. Structural similarity can be determined byany suitable technique, preferably using a suitable software program formaking such assessments. Examples of such programs include the MAPSprogram and the TOP program (described in Lu, Protein Data BankQuarterly Newsletter, #78, 10-11 (1996), and Lu, J. Appl. Cryst., 33,176-183 (2000)). The polypeptide and/or prM/E protein desirably exhibittopological diversity in such contexts (e.g., a topical diversity ofless than about 20, preferably less than about 15, and more preferablyless than about 10), or both. However, polypeptides having highstructural diversity can be suitable and even preferred. Such diverse,but functional, peptides can be obtained by recursive sequencerecombination techniques described above. Alternatively, the structureof proteins can be compared using the PROCHECK program (described in,e.g., Laskowski, J. Appl. Cryst., 26, 283-291 (1993)), the MODELLERprogram, or commercially available programs incorporating such features.Alternatively still, structure predictions can be compared by way of asequence comparison using a program such as the PredictProtein server(available at http://dodo.cpmc.columbia.edu/predictprotein/). Additionaltechniques for analyzing protein structure that can be applied todetermine structural similarity are described in, e.g., Yang and Honig,J. Mol. Biol., 301(3), 665-78 (2000), Aronson et al., Protein Sci.,3(10), 1706-11 (1994), Marti-Remon et al., Annu. Rev. Biophys. Biomol.Struct., 29, 291-325 (2000), Halaby et al., Protein Eng., 12(7), 563-71(1999), Basham, Science, 283, 1132 (1999), Johnston et al., Crit. Rev.Biochem. Mol. Biol., 29(J), 1-68 (1994), Moult, Curr. Opin. Biotechnol.,10(6), 583-6 (1999), Benner et al., Science, 274, 1448-49 (1996), andBenner et al., Science, 273, 426-8 (1996), as well as Int'l PatentApplication WO 00/45334.

[0562] Kits of the invention optionally comprise at least one of thefollowing of the invention: (1) an apparatus, system, system component,or apparatus component as described herein; (2) at least one kitcomponent comprising at least one polypeptide, polynucleotide (orfragment of either thereof), vector, antibody, and/or cell of theinvention; one or more cells comprising at least one polypeptide,polynucleotide, vector, and/or antibody of the invention; a cellexpressing a polypeptide of the invention; a composition, pharmaceuticalcomposition, or vaccine composition (composition suitable for use as avaccine in mammals) comprising at least one of any component above(e.g., polypeptide, polynucleotide, vector, antibody, and/or cell of theinvention) or any combination thereof; (3) instructions for practicingany method described herein, including methods of inducing immuneresponse, methods of immunizing, methods of detecting or diagnosing thepresence or one or more antibodies to at least one dengue virus of oneor more serotypes in a biological sample, therapeutic or prophylacticmethods, instructions for using any component identified in (2) or anyvaccine or composition of any such component; and/or instructions foroperating any apparatus, system or component described herein; (4) acontainer for holding said at least one such component or composition,and (5) packaging materials. In a further aspect, the present inventionprovides for the use of any apparatus, component, composition, or kitdescribed above and herein, for the practice of any method or assaydescribed herein, and/or for the use of any apparatus, component,composition, or kit to practice any assay or method described herein.

[0563] The invention provides computers, computer readable media, andintegrated systems comprising character strings corresponding to thesequence information herein for the polypeptides and nucleic acidsherein, including, e.g., those sequences listed herein and varioussilent substitutions and conservative substitutions thereof. Variousmethods and genetic algorithms (GAs) known in the art can be used todetect homology or similarity between different character strings, orcan be used to perform other desirable functions such as to controloutput files, provide the basis for making presentations of informationincluding the sequences and the like. Examples include BLAST, discussedsupra.

[0564] Different types of homology and similarity of various stringencyand length can be detected and recognized in the integrated systemsherein. E.g., many homology determination methods have been designed forcomparative analysis of sequences of biopolymers, for spell-checking inword processing, and for data retrieval from various databases. With anunderstanding of double-helix pair-wise complement interactions among 4principal nucleobases in natural polynucleotides, models that simulateannealing of complementary homologous polynucleotide strings can also beused as a foundation of sequence alignment or other operations typicallyperformed on the character strings corresponding to the sequences herein(e.g., word-processing manipulations, construction of figures comprisingsequence or subsequence character strings, output tables, etc.). Anexample of a software package with GAs for calculating sequencesimilarity is BLAST, which can be adapted to the invention by inputtingcharacter strings corresponding to the sequences herein.

[0565] Similarly, standard desktop applications such as word processingsoftware (e.g., Microsoft Word™ or Corel WordPerfect™) and databasesoftware (e.g., spreadsheet software such as Microsoft Excel™, CorelQuattro Pro™, or database programs such as Microsoft Access™ orParadox™) can be adapted to the present invention by inputting acharacter string corresponding to the polypeptides or polynucleotides ofthe invention or both, or fragments of either. For example, theintegrated systems can include the foregoing software having theappropriate character string information, e.g., used in conjunction witha user interface (e.g., a GUI in a standard operating system such as aWindows, Macintosh or LINUX system) to manipulate strings of characters.As noted, specialized alignment programs such as BLAST can also beincorporated into the systems of the invention for alignment of nucleicacids or proteins (or corresponding character strings).

[0566] Integrated systems for analysis in the present inventiontypically include a digital computer with GA software for aligningsequences, as well as data sets entered into the software systemcomprising any of the sequences described herein. The computer can be,e.g., a PC (Intel x86 or Pentium chip-compatible DOS™, OS2™ WINDOWS™WINDOWS NT™, WINDOWS95™, WINDOWS98™ LINUX based machine, a MACINTOSH™,Power PC, or a UNIX based (e.g., SUN™ work station) machine) or othercommercially common computer which is known to one of skill. Softwarefor aligning or otherwise manipulating sequences is available, or caneasily be constructed by one of skill using a standard programminglanguage such as Visualbasic, Fortran, Basic, Java, or the like.

[0567] Any controller or computer optionally includes a monitor which isoften a cathode ray tube (“CRT”) display, a flat panel display (e.g.,active matrix liquid crystal display, liquid crystal display), orothers. Computer circuitry is often placed in a box which includesnumerous integrated circuit chips, such as a microprocessor, memory,interface circuits, and others. The box also optionally includes a harddisk drive, a floppy disk drive, a high capacity removable drive such asa writeable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser and for user selection of sequences to be compared or otherwisemanipulated in the relevant computer system.

[0568] The computer may include appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of the fluid direction and transportcontroller to carry out the desired operation. The software can alsoinclude output elements for controlling nucleic acid synthesis (e.g.,based upon a sequence or an alignment of a sequence herein) or otheroperations which occur downstream from an alignment or other operationperformed using a character string corresponding to a sequence herein.

[0569] In a particular aspect of such an embodiment of the invention,the invention provides a computer or computer readable medium comprisinga database comprising a sequence record comprising one or more characterstring corresponding to a nucleic acid sequence selected from the groupof SEQ ID NOS:156-218, 235, 254-271, 285-330, 342, and 344, or apolypeptide sequence selected from the group of SEQ ID NOS:1-49, 65-116,139-148, 153-155, 236-253, 343, and 345.

[0570] The invention provides an integrated system comprising a computeror computer readable medium comprising a database comprising at leastone sequence record, each comprising at least one character stringcorresponding to a nucleic acid or protein sequence selected from any ofSEQ ID NOS:1-49, 65-116, 139-148, 153-218, 235-253, 254-271, 285-330,342-345, the integrated system further comprising a user input interfaceallowing a user to selectively view one or more sequence records. Forsome such integrated systems, the computer or computer readable mediumcomprising an alignment instruction set which aligns the characterstrings with at least one additional character string corresponding to anucleic acid or protein sequence. The instruction set may comprise oneor more of: a local homology comparison determination, a homologyalignment determination, a search for similarity determination, and aBLAST determination. Some such systems may also comprise a user readableoutput element that displays an alignment produced by the alignmentinstruction set.

[0571] In some aspects, the computer or computer readable medium furthercomprises an instruction set which translates at least one nucleic acidsequence comprising a sequence selected from the group of SEQ IDNOS:156-218, 235, 254-271, 285-330, 342, and 344 into an amino acidsequence. In other aspects, the computer or computer readable mediumfurther comprising an instruction set for reverse-translating at leastone amino acid sequence comprising a sequence selected from SEQ IDNOS:1-49, 65-116, 139-148, 153-155, 236-253, 343, and 345 into a nucleicacid sequence. For some such systems, the instruction set selects thenucleic acid sequence by applying a codon usage instruction set or aninstruction set which determines sequence identity to a test nucleicacid sequence.

[0572] Also provided is a method of using a computer system to presentinformation pertaining to at least one of a plurality of sequencerecords stored in a database, each of said sequence records eachcomprising at least one character string corresponding to one or more ofSEQ ID NOS:1-49, 65-116, 139-148, 153-218, 235-253, 254-271, 285-330,342-345, the method comprising: determining a list of one or morecharacter strings corresponding to one or more of said above-referencedSEQ ID NOS, or a subsequence thereof; determining which one or morecharacter strings of said list are selected by a user; and displayingthe selected character strings, or aligning the selected characterstrings with an additional character string. Some such methods furthercomprise displaying an alignment of the selected character string withthe additional character string and/or displaying the list.

[0573] In a further aspect, the invention provides a method ofgenerating and/or selecting a polypeptide variant comprising using acharacter string corresponding to at least one of SEQ ID NOS: 1-49,65-116, 139-148, 153-155, 236-253, 343, and 345, preferably incombination with additional biological functional information (e.g.,neutralizing antibody titer against one or more dengue virus serotypes)to an algorithm, preferably facilitated by a computer media, theanalyzed outcome of which generates one or more amino acid sequences,pattern of sequence characteristics, sequence changes (mutations),and/or structures that are suggestive of a polypeptide that exhibits asimilar and/or improved biological property of such polypeptides. Themethod can include subjecting the character strings to any suitable typeof genetic modeling or algorithm known in the art, including, e.g.,statistical analysis techniques such as Markov modeling, principalcomponent analysis, neural network analysis, randomrecombination-modeling approaches, and physical recombinationapproaches. Examples of such techniques are described in, e.g.,International Patent Applications WO 01/83559, WO 99/49893, and WO01/61344, and U.S. Pat. No. 6,269,312. Additional techniques andprinciples are described in, e.g., International Patent Applications WO00/42561, WO 01/51663, and WO 01/90197 as well as Norton et al. VirusRes 55(1):37-48 (1998), Rappuoli, Curr Opin Microbiol 3(5):445-450(2000), Petersen et al., Scand J. Immunol 53(4):357-364 (2001), andNakai J. Struct Biol 134(2-3):103-116 (2001). Amino acid and nucleotidesequences produced having a non-wild-type sequence generated by such insilico modeling using a character string corresponding to SEQ IDNOS:1-49, 65-116, 139-148, 153-218, 235-253, 254-271, 285-330, 342-345are a feature of the invention.

[0574] Any of the above described features of the polypeptides,polynucleotides, vectors, cells, compositions, and methods of theinvention can be combined in any suitable manner, unless otherwisestated or clearly contradicted by context.

[0575] Any molecule of the invention, including any nucleic acid,polypeptide, protein, peptide, or fusion protein of the invention, orany vector, cell, or composition comprising any such molecule asdescribed herein, can be used in any of the methods and applications ofthe invention described herein. In one aspect, the invention providesfor the use of any such molecule, including any nucleic acid,polypeptide, protein, peptide, or fusion protein, or any vector, cell,or composition comprising any such molecule as described herein, as amedicament, drug, therapeutic or prophylactic agent, or vaccine, for thetreatment or prevention of a disease or disorder, including thosediseases and disorders described herein (e.g., those related to denguevirus infection), or the like. In another aspect, the invention providesfor the use of any such molecule (e.g., any nucleic acid, polypeptide,protein, fusion protein, or peptide of the invention) or any vector,cell, or composition comprising any such molecule, for the manufactureof a medicament, prophylactic or therapeutic agent, drug, or vaccine,for use in any applicable therapeutic or prophylactic method for thetreatment or prevention of a disease or disorder, including thosedescribed herein (e.g., those related to dengue virus infection).

EXAMPLES

[0576] The following examples further illustrate the invention, butshould not be construed as in any way limiting its scope in any way.

Example 1

[0577] This example illustrates the generation and identification ofnovel nucleic acids that encode recombinant, synthetic or mutant denguevirus antigens, polypeptides comprising such dengue virus antigens, theconstruction of an exemplary DNA vectors for delivery of such nucleicacids to mammalian cells, and the expression of such nucleic acids bytransfection of mammalian cells with such a vector, resulting in highlevels of expression and secretion of the encoded dengue antigens.

[0578] A. Synthesis of Novel Dengue Antigen-Encoding NucleotideSequences

[0579] The amino acid sequences of the various proteins of each of thefour WT DEN-1, DEN-2, DEN-3, and DEN-4 viruses were analyzed to identifythe following regions of the polyprotein of each of the four WT denguevirus serotypes: (1) the amino acid segment or fragment of the denguepolyprotein sequence corresponding to the C-terminal 15 amino acids ofthe prM protein (“PRM15”), which 15 amino acid segment of the prMprotein may serve as a signal sequence for the dengue envelope (E)protein; (2) the amino acids comprising most of the full length Eprotein sequence of the dengue polyprotein sequence, i.e., excludingthose amino acid residues corresponding to approximately 5% to about 12%of the C-terminal region of the E protein sequence that encode a portionof the hydrophobic region). Thus, an amino acid sequence was identifiedfrom the polyprotein sequence of each of the four WT dengue virusserotypes; each such amino acid sequence comprised a fusion proteincomprising the C-terminal 15 amino acid residues of the prM protein(e.g., 15 amino acid residues of the M protein) and approximately 90-95%of the N-terminal amino acid residues of the E protein (hence, a termeda “truncated” E protein). See FIG. 14. These amino acid sequencesidentified from each of the wild-type DEN-1, DEN-2, DEN-3, and DEN-4polyproteins were termed Den-1PRM15/trunc E (or Den-1PRM15/tE) (SEQ IDNO:149), Den-2PRM15/trunc E (or Den-2PRM15/tE) (SEQ ID NO:150),Den-3PRM15/trunc E (or Den-3PRM15/tE) (SEQ ID NO:151), andDen-4PRM15/trunc E (or Den-4PRM15/tE) (SEQ ID NO:152) dengue antigenfusion proteins, respectively. Collectively, these sequences may betermed the “truncated” parental polypeptides. These fusion proteins canbe made by expression from plasmid vectors, such as E. coli vectors,viral vectors, baculovirus vectors, or other plasmid vectors, andproduced in insect cells, E. coli cells, or mammalian cell cultures bystandard techniques known in the art and described herein.Alternatively, proteins can be assembled from protein fragments orpeptide fragments made by standard protein synthesis techniques wellknown in the art and discussed above.

[0580] Each of these four identified amino acid sequences was backtranslated to a nucleotide sequence, using a standard human codonfrequency table(http://www.kazusa.orp/codon/cgi-bin/showcodon.cgi?species=Homo+sapiens+[gbpri]),to obtain a human codon optimized DNA sequence.

[0581] DNA oligonucleotides comprising overlapping portions of eachidentified human codon optimized PRM15 and truncated E fusionprotein-encoding sequence were synthesized using standard techniques foreach of the 4 dengue virus serotypes. For each of the virus serotypes,the overlapping oligos that collectively made up the PRM15/E truncatedprotein-encoding sequence were permitted to hybridize. DNA sequencescorresponding to the codon optimized sequences were produced by standardPCR gene synthesis using the hybridized oligos as templates. For each ofthe PCR gene synthesis reactions, a 5′ forward primer comprisingnucleotide sequences overlapping the PRM15 sequence, an additional BamHIsite and a 5′-ACC-3′ Kozak consensus sequence (Cell 15:1109-23 (1978))and a 3′ reverse primer comprising nucleotide sequences overlapping thetruncated E gene and a EcoRI restriction site were used, such that thesesites and the consensus sequence were added to the resultingPRM15/truncated E-encoding PCR products. The resulting human codonoptimized PRM15/truncated E-encoding DNA sequences were designatedDen-1PRM15/truncE CO (or Den-1PRM15/tE CO) (SEQ ID NO:211),Den-2PRM15/truncE CO (or Den-2PRM15/tE CO) (SEQ ID NO:212),Den-3PRM15/truncE CO (or Den-3PRM15/tE CO (SEQ ID NO:213), andDen-4PRM15/truncE CO (or Den-4PRM15/tE CO) (SEQ ID NO:214),respectively, where “PRM15” refers to a signal nucleotide sequencecomprising a sequence encoding the 15 amino acids of the C terminus ofprM (i.e., PRM15) and typically an additional methionine residue (whichis the first amino acid residue before the 15 amino acid sequence),“trunc” E or “tE” refers to the nucleotide sequence encoding a truncatedE protein, and “CO” refers to codon optimized. Collectively, thesenucleotide sequences may be termed the truncated parental nucleotidesequences, since they were used as parental nucleic acids in therecursive sequence recombination methods described herein.

[0582] Similar procedures are used to identify and make a polypeptidesequence comprising: the 15 amino acid residues of the capsid (C)protein of a particular WT dengue virus serotype polyprotein (e.g.,Den-1, Den-2, Den-3, or Den-4), the amino acid sequence corresponding tothe full length prM protein of said WT dengue virus polyprotein, and theamino acid sequence corresponding to said full length E protein of theWT dengue virus polyprotein. Such procedures are conducted using each ofthe four WT dengue virus polyproteins, and the resulting polypeptidesequences are termed Den-1 C15/full prM/full E (or Den-1 C15/prM/E) (SEQID NO:227), Den-2 C15/full prM/full E (or Den-2 C15/prM/E) (SEQ IDNO:228), Den-3 C15/full prM/full E (or Den-3 C15/prM/E) (SEQ ID NO:229),Den-4 C15/full prM/full E (or Den-4 C15/prM/E) (SEQ ID NO:230) dengueantigen fusion proteins, respectively.

[0583] Human codon optimized sequences that encode each of these dengueantigen fusion proteins are determined and made as described above, andthe resulting nucleotide sequences are termed: Den-I C₁₅/full prM/full ECO (or Den-1 C15/prM/E CO) (SEQ ID NO:231), Den-2 C15/full prM/full E CO(or Den-2 C15/prM/E CO) (SEQ ID NO:232), Den-3 C15/full prM/full E CO(or Den-3 C15/prM/E CO) (SEQ ID NO:233), and Den-4 C15/full prM/full ECO (or Den-4 C15/prM/E CO) (SEQ ID NO:234) dengue antigen nucleotidesequences, respectively.

[0584] B. Construction of pMaxVax10.1

[0585] An exemplary mammalian expression vector termed “pMaxVax10.1”(see FIG. 1) comprises, among other things: (1) a promoter for drivingthe expression of a transgene (or other nucleotide sequence) in amammalian cell (including, e.g., but not limited to, a CMV promoter or avariant thereof, and shuffled, synthetic, or recombinant promoters,including those described in PCT application having InternationalPublication No. WO 02/00897; (2) a polylinker for cloning of one or moretransgenes (or other nucleotide sequence); (3) a polyadenylation signal(e.g., polyA sequence); and (4) a prokaryotic replication origin andantibiotic resistant gene for amplification in E. coli. The constructionof the vector is briefly described herein, although several suitablealternative techniques are available to produce such a DNA vector (e.g.,applying the principles described elsewhere herein).

[0586] In one embodiment, the minimal plasmid Col/Kana comprises thereplication origin ColE1 and the kanamycin resistance gene (Kana^(r)).The ColE1 origin of replication (ori) mediates high copy number plasmidamplification.

[0587] In one embodiment, the ColE1 ori was isolated from vector pUC19(New England Biolabs, Inc.) by application of standard PCR techniques.To link the ColE1 origin to the Kana^(r)gene, NgoMIV (or “NgoMI”) andDraIII recognition sequences were added to the 5′ and 3′ PCR primers,respectively. NgoMIV and DraIII are unique cloning sites in pMaxVax10.1.For subsequent cloning of the mammalian transcription unit, the 5′forward primer also was designed to include the additional restrictionsite NheI downstream of the NgoMIV site and EcoRV and BsrGI cloningsites upstream of the DraIII site the 3′ reverse primer. All of theprimers were designed to include additional 6-8 base pairs overhang foroptimal restriction digest. Specifically, the sequence for the 5′forward primer (“pMaxVax primer 1”) isacacatagcgccggcgctagctgagcaaaaggccagcaaaaggcca (SEQ ID NO:331) and thesequence for the 3′ reverse primer (“pMaxVax primer 2”)aactctgtgagacaacagtcataaatgtacagatatcagaccaagtttactcatatatac (SEQ IDNO:332).

[0588] Typically, the ColE1 PCR reactions were performed withproof-reading polymerases, such as Tth (PE Applied Biosystems), Pfu,PfuTurbo and Herculase (Stratagene), or Pwo (Roche), under conditions inaccordance with the manufacturer's recommendations. By way ofillustration, a typical Herculase polymerase PCR reaction contains 1 μltemplate plasmid DNA (1-10 ng/μl), 5 μl 10× buffer, 1 μl dNTPs(deoxynucleotide triphosphates) at 10 mM each, 1 μl forward primer (20μM), 1 μl reverse primer (20 μM), 40 μl deionized, sterile water and 0.5μl Herculase polymerase in a 50 μL reaction. Such PCR reactions wereperformed at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C.for 30 seconds per cycle, for a total of 25 cycles.

[0589] The ColE1 PCR product was purified with phenol/chloroform usingPhase lock Gel™Tube (Eppendorf) followed by standard ethanolprecipitation. The purified ColE1 PCR product was digested with therestriction enzymes NgoMIV and DraIII according to the manufacturer'srecommendations (New England Biolabs, Inc.) and gel purified using theQiaExII gel extraction kit (Qiagen) according to the manufacturer'sinstructions.

[0590] In this embodiment, the Kanamycin resistance gene (transposonTn903) was isolated from plasmid pACYC177 (New England Biolabs, Inc.)using standard PCR techniques. Specifically, a 5′ PCR primer (“pMaxVaxprimer 3”), ggcttctcacagagtggcgcgccgtgtctcaaaatctct (SEQ ID NO:333),comprising sequences homologous to the 5′ kanamycin gene and anadditional DraIII site upstream of an AscI site, and a 3′ primer(“pMaxVax primer 4”), ttgctcagctagcgccggcgccgtcccgtcaagtcagcgt (SEQ IDNO:334), comprising sequences homologous to the 3′ kanamycin gene and aNgoMIV cloning site, were used to amplify the Kana^(r) gene frompACYC177. The PCR reactions, product purification and digest with DraIIIand NgoMIV were performed as described above. About 20 ng of each of theKanar PCR product and ColE1 PCR product were obtained and ligated in a20 μl reaction, containing 2 μl 10× buffer and 1U ligase (Roche).Amplification in E. coli was performed using standard procedures asdescribed in Sambrook, supra. Plasmids were purified with theQiaPrep-spin Miniprep kit (Qiagen) following the manufacturer'sinstructions and digested with BsrG1 and DraIII for subsequent ligationof the mammalian transcription unit (promoter and polyA).

[0591] In one embodiment, the pMaxVax10.1 vector comprise a CMVimmediate early enhancer promoter (CMV IE), which was isolated from DNAof the CMV virus, Towne strain, by standard PCR methods. The cloningsites EcoRI and BamHI were incorporated into the PCR forward and reverseprimers. The EcoRI and BamHI digested CMV IE PCR fragment was clonedinto pUC19 for amplification. The CMV promoter was isolated from theamplified pUC19 plasmid by restriction digest with BamHI and BsrGI. TheBsrGI site is located 168 bp downstream of the 5′ end of the CMVpromoter, resulting in a 1596 bp fragment, which was isolated bystandard gel purification techniques for subsequent ligation.

[0592] In one embodiment, a polyadenylation signal from the bovinegrowth hormone (BGH) gene was used. Other polyadenylation signals (e.g.,SV40 poly A sequences) may also be employed. In this instance, a BGHnucleotide sequence was isolated from the pcDNA3.1 vector (Invitrogen)by standard PCR techniques. Briefly, a 5′ PCR forward primer (“pMaxVaxprimer 5”), agatctgtttaaaccgctgatcagcctcgactgtgccttc (SEQ ID NO:335),which includes recognition sites for the restriction enzymes PmeI andBglII to form part of the p.MaxVax10.1 vector polylinker, and a 3′reverse primer (“pMaxVax primer 6”),acctctaaccactctgtgagaagccatagagcccaccgca (SEQ ID NO:336), which includesa DraIII site for cloning to the minimal plasmid Col/Kana, were preparedby standard techniques and used to amplify a BGH polyA PCR product. TheBGH polyA PCR product was diluted 1:100. 1 μl of the diluted BGH polyAPCR product was used as a template for a second PCR amplification usingthe same 3′ reverse primer and a second 5′ primer (“pMaxVax primer 7”),ggatccggtacctctagagaattcggcggccgcagatctgtttaaaccgctga (SEQ ID NO:337),which overlapped the 5′ end of the template by 20 bp, and containedanother 40 bp 5′ sequence comprising BamHI, KpnI, XbaI, EcoRI, and NotIrestriction sites for inclusion of these sites in the p.MaxVax10.1vector polylinker.

[0593] The final ligation reaction to form pMaxVax10.1 was performedwith about 20 ng each of the BsrG1 and BamHI digested CMV IE PCRproduct, BamHI and DraIII digested polylinker and BGH poly A PCRproduct, and the DraIII and BsrG1 digested minimal plasmid Col/Kana in a50 μl reaction with 5 μl 10× ligase buffer and 2U ligase (Roche).Ligation, amplification and plasmid purification were performed asdescribed above. The plasmid was transfected into E. coli using standardtechniques for cloning.

[0594] C. Construction of pMaxVax10.1 Dengue Virus Antigen ExpressionVector

[0595] In one aspect, BamHI and EcoRI digested and gel-purifiedDen-1PRM15/tE CO, Den-2PRM15/tE CO, Den-3PRM15/tE CO, and Den-4PRM15/tECO nucleic acids (described above) were each cloned into the pMaxVax10.1vector by digesting the vector with BamHI and EcoRI using standardtechniques, gel purifying the linearized vector, and ligating therespective dengue antigen-encoding sequences (separately in 4 differentligation reactions to the vector to form 4 plasmid vector constructs,each comprising a codon optimized nucleic acid encoding Den-1PRM15/tE,Den-2PRM15/tE, Den-3PRM15/tE, or Den-4PRM15/tE fusion protein antigen(see FIG. 2). Such vectors are typically termedpMaxVax10.1_(Den-1PRM15/tE CO), pMaxVax10.1_(Den-2PRM15/tE CO),pMaxVax10.1_(Den-3PRM15/tE CO), and pMaxVax10.1_(Den-4PRM15/tE CO),respectively. FIG. 2 shows an exemplary pMaxVax10.1 expression vectorcomprising a nucleotide sequence corresponding to Den-2PRM15/tE CO.These dengue antigen fusion protein-encoding expression vectors werepropagated in E. coli using standard techniques to obtain a populationof plasmids for transfection experiments.

[0596] Similar procedures are used to construct pMaxVax10.1 expressionvectors comprising each of the nucleotide sequences described above:Den-1 C15/full prM/full E CO (SEQ ID NO:231), Den-2 C15/full prM/full ECO (SEQ ID NO:232), Den-3 C15/full prM/full E CO (SEQ ID NO:233), andDen-4 C15/full prM/full E CO (SEQ ID NO:234).

[0597] D. Dengue Antigen Expression and Secretion Analysis for Den-1PRM15/tE CO, Den-2PRM15/tE CO, Den-3PRM15/tE CO, and Den-4PRM15/tE CO

[0598] Dengue antigens encoded by wild-type dengue nucleic acids (orportions or fragments thereof), are typically poorly expressed andsecreted. To assess the expression and/or secretion of dengue antigensexpressed from the above-described pMaxVax10.1 expression vectors thefollowing techniques were performed.

[0599] Populations of human 293-HEK cells were grown in tissue cultureunder standard conditions, and each population was transfected with apMaxVax10.1 expression vector comprising one of the nucleic acidsequences described above: Den-1PRM15/tE CO, Den-2PRM15/tE CO,Den-3PRM15/tE CO, or Den-4PRM15/tE CO. Transfections were carried outusing commercially available transfections reagents, typically Effectene(Qiagen) and FuGene (Boehringer/Roche), in accordance with themanufacturer's instructions (e.g., with respect to amount of plasmidused for transfection). Each population of transfected 293 cells wasincubated for about 48-72 hours under conditions permissive fortransgene expression.

[0600] Two sets of samples of each of the transfected 293 cell cultureswere prepared. The first set of samples was prepared by harvesting thesupernatants of each population of cells transfected with one of the 4expression plasmids and concentrating the volume 10-20 fold by standardmethods (e.g., centrifugation using Ultrafree-4 centrifugal filter,Millipore, Mass.). The protein secretion levels were determined byWestern blot analysis. In the other set of samples, cell of eachpopulation of cells transfected with one of the 4 expression plasmidswere harvested, subjected to cell lysis, and the resulting lysatecollected and subjected to Western blot analysis to determine expressionlevels.

[0601] For Western blot analysis, the proteins from the cell lysates andcell supernatants, respectively, were separated by polyacrylamide gelelectrophoresis and blotted to nitrocellulose membranes using thetechnique described by the manufacturers (NuPage™ and Invitrogen,respectively). Cell lysates were run in one lane (marked “L”) and cellsupernatants were run in another lane (marked “SN”) on a singlepolyacrylamide gel for each nucleic acid tested (e.g.,p.MaxVax10.1_(Den-2PRM15/tE Co) transfected cell lysates andsupernatants were run on a single gel).

[0602] Polyclonal anti-DEN-1 mouse antisera and anti-DEN-3 mouseantisera were obtained from ATCC, polyclonal anti-DEN-4 mouse antiserawere received from the WHO antibody collection, and a monoclonalanti-DEN-2 antibody was received from United States Biological(Swampscott Mass.—USA) (polyclonal anti-DEN-2 mouse antisera repeatedlyfailed to bind to denatured wild-type DEN-2 antigens on filters and,accordingly, were not used).

[0603] The filters were incubated with dengue virus serotype specificantibodies corresponding to the type of dengue antigen encoded by thenucleic acid used to transfect the samples (i.e., anti-DEN-2 mAb wasused for filters blotted with the lysates or supernatant frompMaxVax10.1_(Den-2PRM15/tE CO) transfected cells). The antibody-antigenincubations were performed for 1 hour at room temperature, the filterswere washed 5 times for 25 minutes with PBS Buffer and 0.1% Tween 20,and the filters were further incubated with a secondaryenzyme-conjugated (either a horse radish peroxidase (HRP)-conjugated oralkaline phosphatase-conjugated) anti-mouse antibody. After a 1-hourincubation at room temperature, the filters were washed and incubatedwith the enzyme substrates for colorimetric detection. The Western blotobtained by this experiment is shown in FIG. 3.

[0604] The Western blot shown in FIG. 3 demonstrates that antigensencoded by Den-1PRM15/tE CO, Den-2PRM15/tE CO, Den-3PRM15/tE CO, andDen-4PRM15/tE CO were well expressed in transfected 293 cells. However,only antigens expressed from pMaxVax10.1 vectors comprisingDen-2PRM15/tE CO, Den-3PRM15/tE CO, and Den-4PRM15/tE CO appear to besecreted from transfected cells. In comparison, truncated envelopeantigens expressed from wild-type dengue virus nucleotide sequences havebeen shown to be poorly expressed and very poorly secreted in mammaliancells.

[0605] These experiments demonstrate that nucleic acid sequencescomprising Den-1PRM15/tE CO, Den-2PRM15/tE CO, Den-3PRM15/tE CO, andDen-4PRM15/tE CO sequences are capable of high levels of dengue antigenexpression in transfected cells. Moreover, the results of theseexperiments demonstrate that select nucleic acid sequences of theinvention can be used to effectively express dengue antigens as secretedproteins.

[0606] Similar methods are used to assess the expression and/orsecretion of pMaxVax10.1 expression vectors comprising each of thefollowing nucleotide sequences described above: Den-1 C15/full prM/fullE CO (SEQ ID NO:231), Den-2 C15/full prM/full E CO (SEQ ID NO:232),Den-3 C15/full prM/full E CO (SEQ ID NO:233), and Den-4 C15/fullprM/full E CO (SEQ ID NO:234).

Example 2

[0607] This example illustrates exemplary methods for generatinglibraries of nucleic acids encoding recombinant dengue antigens andscreening or selecting from such libraries to identify recombinantnucleic acids that encode recombinant dengue antigens in mammaliancells.

[0608] Libraries of recombinant nucleic acid sequences were generated byrecursive sequence recombination procedures using purified Den-1PRM15/tECO, Den-2PRM15/tE CO, Den-3PRM15/tE CO, and Den-4PRM15/tE CO, e.g., asparental nucleotide sequences. The recursive sequence recombinationprocedures were conducted as described previously in, e.g., Stemmer(1994) Nature 370:389-391 and Crameri, A. et al. (1998) Nature391:288-91, U.S. Pat. No. 5,837,458, and other references cited above inthe section describing recursive sequence recombination, eachincorporated herein by reference in its entirety for all purposes.

[0609] Briefly, each pMaxVax10.1 expression vector comprising a Den-1PRM15/tE CO, Den-2 PRM15/tE CO, Den-3 PRM15/tE CO, or Den-4 PRM15/tE COnucleotide sequence was cleaved by restriction enzymes flanking thedengue PRM15/tE nucleotide sequence, and each resulting nucleotidefragment were isolated by standard gel purification and subjected torecursive sequence recombination. The recombined nucleic acid productswere then amplified by PCR with rescue primers located upstream of the5′ BamHI and 3′ of the EcoRI cloning sites, digested with BamHI andEcoRI, and gel purified using standard techniques. The isolatedrecombinant nucleic acids were ligated into pMaxVax10.1 vectors (e.g.,as described in Example 1 to form a recombinant library of recombinantDNA plasmid vectors, which were subsequently cloned in E. coli usingstandard library transfection techniques (see, e.g., Sambrook, supra)and according to manufacturer's instructions.

[0610] The transformed cells were plated overnight, the individualcolonies were pooled, and plasmid DNA was prepared by standardpurification methods (Qiagen). Plasmid DNA of the pMaxVax10.1 expressionlibraries was transfected into HEK-293 cells in culture using Superfectreagent (Qiagen) according to the manufacturer's instructions. Severallibraries so produced were then analyzed for dengue antigen expressionby flow cytometry (Fluorescence-Activated Cell Sorter—FACS analysis)with anti-dengue virus antibodies from mouse ascitic fluid.

[0611] Specifically, to perform FACS analysis, about 1×10⁵ transfected293 cells were incubated with a mixture of the mouse anti-dengue virusantibodies (as described above), dissolved in PBS buffer containing 2%fetal calf serum (FCS). The optimal antibody dilution was evaluated on acase-by-case basis for each antibody. In general, serial test dilutionsof 1:500, 1:1000, and 1:2000 were used. The cells were stained for 30minutes on ice and washed 3 times with PBS buffer before being incubatedwith appropriate secondary antibodies, which were coupled with afluorescent detection reagent (goat anti-mouse phycoerythrin conjugate,CalTag Lab). The staining concentration was determined for each labeledantibody to provide a maximal Mean Fluorescence Intensity (MFI) andminimal background signal (e.g., optimum staining concentration was theconcentration per 10⁵ cells). After 30 minutes incubation on ice, thecells were washed 3 times with PBS and analyzed by FACS. Specifically,cells were analyzed using a FACSCalibur flow cytometer and CellQuestsoftware (BDIS, San Jose, Calif.).

[0612] First round libraries of recombinant nucleic acids were preparedaccording to this protocol. Recombinant nucleic acids encoding denguevirus PRM15/tE fusion proteins cloned into pMaxVax vectors, wherein suchnucleic acid library-transfected cells were incubated with a humandengue virus antisera, were transfected into human 293 cell cultures andanalyzed by standard FACS analyses, as described above, using a programthat provides a graphical output of the number of positive cells againstfluorescence intensity (software settings and dyes were selected suchthat dead cells were excluded from the output signal as were aggregatedcell masses). The recombinant nucleic acids were produced by recursivelyrecombining the following codon optimized dengue virus antigen parentalnucleotide sequences with one another: Den-1PRM15-/tE CO, Den-2PRM15/tECO, Den-3PRM15/tE CO, and Den-4PRM15/tE CO. Each library of recombinantPRM15/tE-encoding dengue virus nucleic acids comprised at least onerecombinant nucleic acid that encoded a recombinant PRM15/tE denguevirus polypeptide that was bound by one or more murine anti-dengue virusantibodies. The experiments also included a population of 293 cellstransfected with a pMaxVax10.1_(null) vector (FIG. 1) lacking any denguevirus nucleic acid insertion (termed a “null vector”) as a control (C)(data not shown).

[0613] Similar experiments were repeated with dengue virus antiseraobtained from human patients (Immunology Consultants LaboratoryInc.—Sherwood, Oreg.) infected with dengue viruses (of unknown virusserotype(s)) (data no shown). Each library of recombinant PRM15/tEdengue virus nucleic acids also comprised at least one recombinantdengue virus nucleic acid that encoded a PRM15/tE dengue viruspolypeptide that was bound by one or more human anti-dengue virusantisera.

[0614] To isolate such individual recombinant nucleic acids, individualE. coli colonies were picked from the plated libraries and inoculatedinto 96-well blocks containing 1.2 ml Terrific Broth-amp (50 μg/ml). The96-well plate cultures were grown for 20 hours at 37° C., and plasmidDNA was purified using the Biorobot (Qiagen, Valencia, Calif.). HEK-293cells were plated in 96-well plates at a density of 2×10⁴ cells per wellthe day prior to transfections. The cells were transfected withindividual recombinant pMaxVax10.1 vectors, each comprising arecombinant dengue virus nucleic acid, and a pMaxVax10.1_(null) “nullvector” as a control (which lacked a nucleic acid encoding a recombinantor WT dengue virus PRM15/tE), using a Superfect (Qiagen) transfectionsystem according to the manufacturer's instructions. After about 48hours incubation under conditions permissive for transgene expression,the transfected 293 cells were harvested or lysed using standardtechniques, depending on the type of analysis to be performed on thecell or aspirated cell-free cell medium (supernatant) (e.g., whether thecells were subjected to FACS or Western blot analysis, as describedfurther below).

[0615] Such techniques were used to screen or select from the librariesof recombinant polynucleotides produced by the above-described method toidentify cells that comprised nucleic acids encoding polypeptides thatreacted with specific dengue virus antibodies. Briefly, each well wasanalyzed with anti-dengue virus antibodies from mouse ascitic fluidagainst all four virus serotypes by FACS, as described above. Positivecells were counted in the FL2-H channel and graphically plotted. Thegraphical output obtained from these experiments was compared to theoutput obtained from similar experiments performed with the vectorcontrol (null vector). Cells that exhibited an intensity of at leastabout 10² were considered positive for recombinant PRM15/tE dengue virusAg expression (data not shown).

[0616] Plasmid DNAs of pMaxVax10.1 DNA vectors corresponding to positiveclones identified in such 96-well high throughput screening assays wereagain transfected into 293 cells, and these cells were harvested andanalyzed for tetravalent antigen expression against with the fourserotype specific antibodies, as described below (Example 3).

[0617] The results of these experiments demonstrate that recursivesequence recombination and appropriately devised selection/screeningprocedures can be applied to DEN-1, DEN-2, DEN-3, or DEN-4 PRM15/tE CO(codon-optimized) antigen-encoding nucleic acids to generate librariesof novel recombinant nucleic acids and identify therefrom specificrecombinant nucleic acids comprising nucleotide sequences that encoderecombinant dengue antigens that reacted with murine and/or human denguevirus antibodies.

Example 3

[0618] This example describes the production and identification ofrecombinant nucleic acids that encode multivalent dengue antigens usingrecursive sequence recombination methods and chosen selection/screeningprocedures.

[0619] Clone 2/7 was selected from a library of clones comprisingrecombinant nucleic acids produced according to Example 2, after beingidentified as positive for the expression of a recombinant dengueantigen by FACS analysis. The nucleic acid sequence of clone 2/7,comprising a recombinant PRM15/trunE sequence is shown in SEQ ID NO:156.(The nucleic acid sequence of clone 2/7 comprising only the recombinanttruncE nucleotide segment is shown in SEQ ID NO:285.)

[0620] 293 cells were transfected with pMaxVax10.1_(2/7) orpMaxVax10.1_(Den-3PRM15/tE CO) using standard techniques and accordingto manufacturer's instructions, as described above. The transfectedcells were cultured for about 48 hours under conditions permissive fornucleotide (e.g., transgene) expression, harvested, and divided intofour for separate staining reactions. Serotype specific mouse DEN-1,DEN-2, DEN-3, and DEN-4 polyclonal antisera were then added topMaxVax10.1_(2/7) or pMaxVax10.1_(Den-3PRM15/tE CO) transfected cells,followed by an incubation with appropriately labeled secondaryantibodies, and the cells subjected to FACS analyses, as described inExample 2. The results of these experiments, which are shown in FIG. 4,demonstrate that the cloned recombinant nucleic acid of E. coli clone2/7 (clone 2/7—SEQ ID NO:156), which encodes a recombinant antigen (SEQID NO:65) that is expressed on the surface of mammalian cells, isreactive with antibodies of all 4 dengue virus serotypes (i.e.,tetravalent).

[0621] In comparison, Den-3PRM15/tE CO expressed an antigen having a WTDEN-3PRM15/truncated E fusion protein sequence (SEQ ID NO:151) that wasreactive only with antibodies against DEN-3 and also cross-reactive withanti-DEN-1 antibodies.

[0622] These experimental results demonstrate the production of cellsurface recombinant antigens that are cross-reactive with antibodies toall 4 serotypes of dengue viruses by one of the inventive methods of theinvention. Moreover, the experiment illustrates an effective techniquefor identifying recombinant nucleic acids encoding multivalent antigensfrom a recombinant nucleic acid library produced according to themethods described herein.

Example 4

[0623] This example illustrates the identification of recombinantmultivalent dengue antigens produced according to the methods describedherein by Western blot analyses.

[0624] Six representative “PMR15/tE” clones (designated 2/7, 5/21, 2G11,6E12, 6A11, and 6D8) identified as positive for the expression ofrecombinant dengue antigens by the methods described in Example 2 wereselected. The recombinant nucleic acid-containing pMaxVax10.1 vectorscorresponding to each of these 6 clones were isolated, as describedabove. Eleven cultures of 293 cells were prepared. Six cultures weretransfected individually with one of the six pMaxVax10.1 vectorscomprising a recombinant dengue PRM15/tE nucleotide sequence. Each ofthe remaining five cultures was transfected individually with one of thefollowing vectors comprising a PRM15/tE CO parental nucleotidesequence—pMaxVax10.1_(Den-1PRM15/tE CO), pMaxVax10.1_(Den-2PRM15/tE CO),pMaxVax10.1_(Den-3PRM15/tE CO), pMaxVax10.1_(Den-4PRM15/tE CO)(collectively referred to as “parental sequences” or “parentsequences”), pMaxVax10.1_(null) (vector control; no antigen-encodingsequence added)—under identical conditions. All transfections wereperformed using standard techniques and in accordance withmanufacturers' instructions.

[0625] After about 48 hours incubation, under conditions permissive fornucleotide (e.g., transgene) expression, the transfected 293 cells wereharvested, lysed and then separately subjected to polyacrylamide gelelectrophoresis and Western blot analysis using mouse DEN-1, DEN-3, andDEN-4 antisera (designated α-DEN-1, α-DEN-3, and α-DEN-4, respectively)and appropriately labeled secondary antibodies, using the techniquesdescribed in Example 1. The results of these experiments are shown inFIG. 5.

[0626]FIG. 5 shows that each of the selected recombinant nucleic acidsencoded a secreted recombinant antigen that reacted with antibodies toDEN-1, DEN-3, and DEN-4. At best, only some of the parental nucleotidesequence-encoded antigens showed minimal cross-reactivity withantibodies other than those with which they are normally associated(see, e.g., the reactivity of Den-1PRM15/tE CO-encoded antigen withDEN-3 antibodies), resulting in a Western blot marked by light bandscompared to the consistently well-defined bands observed with the sixrecombinant dengue antigens. Sequencing analysis determined that 2/7 and6A11 were encoded by identical nucleic acid sequences, as were 5/21 and6D8 explaining the remarkably similar banding patterns observed forthese antigens.

[0627] The results of these experiments demonstrate an inventive methodfor producing and identifying (e.g., screening/selecting) recombinantsecreted dengue antigens (comprising recombinant PRM15/tE fusionproteins or related recombinant truncated E proteins lacking the PRM15sequence) that cross-react with (or bind or specifically bind to)anti-dengue virus antigen antibodies against multiple (e.g., at leasttwo, at least three) dengue virus serotypes in mammalian cells.

[0628] Analogous procedures are used to produce and identify recombinantsecreted dengue antigens comprising, e.g., recombinant C 15/full lengthprM/full length E fusion proteins (or related recombinant full lengthprM/full length E fusion proteins lacking the C15 sequence and theinitial Met residue) that cross-react with (or bind or specifically bindto) anti-dengue virus antigen antibodies against at multiple denguevirus serotypes in subjects.

Example 5

[0629] This example describes methods to produce and identify (e.g.,screening/selecting) recombinant dengue antigens that induce theproduction of antibodies to dengue viruses of multiple virus serotypesin vivo.

[0630] Nucleic acid libraries comprising recombinant polynucleotidesequences were produced using recursive sequence recombinationtechniques as described, e.g., in Example 2. pMaxVax10.1 plasmid vectorscomprising members of the library of recombinant nucleic acid sequences(which encode PRM15/trunE dengue virus antigen fusion proteins) wereconstructed. In one aspect, at least sixteen recombinant antigenscorresponding to 16 clones were identified as positive for theexpression of tetravalent dengue antigens (e.g., dengue antigensreactive with all antibodies against all four serotypes) by FACSanalysis described in Example 2. The pMaxVax10.1 plasmid vectors usedfor transformation of the clones were isolated and purified using thetechniques described above for DNA immunization experiments, and thenused for immunizations in mice as follows.

[0631] Inbred mice (BALB/c) were individually injected with endotoxinfree purified (Qiagen) pMaxVax10.1 vector DNA comprising one of thesixteen recombinant nucleic acids or Den-3PRM15/tE CO. Three mice wereinjected with 50 μg of plasmid DNA of one of the indicated plasmid typesin each leg muscle. Three mice received an identical dose of a controlvector, pMaxVax10.1_(null). All of the immunized mice received boosterimmunizations of identical dosage as the initial DNA immunization at day14 after the initial immunization.

[0632] 40 μl of serum was collected from each immunized mouse 28 daysafter the initial plasmid DNA injection and analyzed for antibodyinduction in ELISA assays. ELISA plates (Nunc Immuno Maxisorp(Roskilde—Denmark)) were coated overnight at 4° C. with the testantigens (inactivated dengue viruses of each serotype—Den-1, Den-2,Den-3, and Den-4—supplied by immunology Consultants Laboratory,Inc.—Sherwood, Oreg.) using standard techniques and according to themanufacturer's instructions. The plates were washed 3 times with PBSbuffer containing 0.1% Tween 20 and blocked with 3% BSA/PBS/0.1% Tween20for 1 hour at 37° C. to reduce unspecific binding. The plates werewashed 3 times with PBS/0.1% Tween 20 and incubated for 1 hour at 37° C.with the anti-dengue test sera in a 1:100 dilution and after additional3 washing steps incubated for 1 hour at 37° C. with the secondaryantibodies (goat anti-mouse HRP conjugates, Amersham) at a 1:3000dilution. The plates were finally washed 5 times with PBS/0.1% Tween 20and incubated with TMB peroxidase substrates (Tetramethyl Benzidine,Pierce). The color reaction was stopped with 2M H₂SO₄, and the opticaldensity (absorbance) for each sample was analyzed spectrophotometricallyat 450 nanometers (nm) on an ELISA plate reader. Alternatively, ELISAassays can be performed by using other standard assay formats,including, e.g., that described in Raviprakash et al., J. Gen. Virology81:1659-1667 (2000), which is incorporated herein by reference in itsentirety for all purposes).

[0633] A least seven of the sixteen recombinant PRM15/tE dengue virusantigen-encoding nucleic acids tested—2G11 (SEQ ID NO:157), 2/7 (alsotermed “6A11”)(SEQ ID NO:156), 6E12 (SEQ ID NO:159), 6C6 (SEQ IDNO:160), 5/21 (also termed “6D8”) (SEQ ID NO:158), 6F4 (SEQ ID NO:161),and 7A9 (SEQ ID NO:162), were identified as encoding seven respectiverecombinant antigens (i.e., 2G11 (SEQ ID NO:66), 2/7 (6A 11)(SEQ IDNO:65), 6E12 (SEQ ID NO:69), 6C6 (SEQ ID NO:68), 5/21 (6D8) (SEQ IDNO:67), 6F4 (SEQ ID NO:70), and 7A9 (SEQ ID NO:71) that, upon expressionin vivo, produced antibodies against DEN-1, DEN-2, DEN-3, and DEN-4 thatwere detected in a standard ELISA assay. The signal peptide sequence istypically cleaved after transport of the tE protein into the ER.

[0634] The average optical density (OD) values for each of these seventetravalent antigens were calculated for each serotype-specific ELISAplate tested. These values were plotted on a graph along with theaverage OD values observed for mice injected withpMaxVax10.1_(Den-3PRM15/tE CO) and pMaxVax10.1_(null) on each of thedengue virus serotype-specific ELISA plates (data not shown).Representative data for four of these recombinant tetravalent antigens,2G11, 2/7, 6E12, 5/21) are shown in FIG. 6 (Example 6). In this figure,pMV refers to pMaxVax10.1_(null).

[0635] In vivo injection of each mouse with a population of pMaxVax10.1DNA plasmid expression vectors, wherein each said population of vectorscomprised one such recombinant dengue antigen-encoding nucleotidesequence, resulted in the production of antibodies that reacted withmultiple serotype-specific dengue antigens in the standard ELISA. Forexample, antisera that were obtained from immunized mice, wherein eachsuch mouse had been injected with a population of pMaxVax10.1 vectors,each of said population of vectors comprising one of the followingrecombinant DNA sequences—2G11 DNA, 2/7 (6A11) DNA, 5/21 (6D8) DNA, or6E12 DNA, produced higher OD levels than antisera that were obtainedfrom mice injected with a population of pMaxVax10.1_(Den-1PRM15/tE CO)expression vectors, and tested on ELISA plates, coated with inactivatedDEN-1 virus. Significantly, sera obtained from mice immunized with anyof the seven of these plasmids, each comprising one of the sevenrecombinant antigen-encoding DNA sequences, exhibited higher OD levelsthan sera obtained from mice immunized with pMaxVax10.1_(Den-2PRM15/tE)when analyzed on inactivated DEN-2 virus coated ELISA plates. Moreover,OD levels for sera obtained from mice immunized with pMaxVax10.1_(2/7)DNA, pMaxVax10.1_(5/21) DNA, pMaxVax10.1_(2G11) DNA, pMaxVax10.1_(6E12)DNA, were also higher than those observed for sera obtained from miceimmunized with pMaxVax10.1_(Den-3PRM15/tE CO) and assayed on inactivatedDEN-3 virus coated ELISA plates. OD levels for the plasmids comprisingthe shuffled DNA-encoded antigens pMaxVax10.1_(2G11) DNA andpMaxVax10.1_(6E12) DNA were at least comparable to those of miceinjected with pMaxVax10.1_(Den-4PRM15/tE) when assayed on inactivatedDEN-4 virus coated ELISA plates.

[0636] The results of this experiment demonstrate the effectiveness ofrecursive sequence recombination and appropriate screening/selectionassays in generating and identifying nucleic acids encoding recombinant(PRM15/tE) dengue antigens that upon expression in vivo induce orpromote the production of antibodies that react with (or bind orspecifically bind to) dengue virus antigens of multiple dengue virusserotypes. The results also demonstrate that recombinant nucleic acidsof the invention (and recombinant antigens encoded therefrom) are usefuland effective in inducing or promoting the production of antibodies thatreact with (or bind or specifically bind to) dengue virus antigens ofmultiple dengue virus serotypes in vivo in subjects, including mammals.

Example 6

[0637] This example illustrates the generation of a library ofrecombinant nucleic acids by a second round of recursive sequencerecombination, and the identification and/or isolation of recombinantnucleic acids encoding recombinant dengue antigens from the librarywhich, when expressed in vivo, induce or enhance the production ofantibodies to dengue viruses of multiple dengue virus serotypes.

[0638] The seven nucleic acids identified as encoding tetravalentantigens in Example 5 (PRM15/trunE format) were isolated, purified, andsubjected to a second round of recursive sequence recombination (e.g.,DNA shuffling and appropriately defined selection/screening) to producea first second round library of recombinant nucleic acids (PRM15/trunEformat), all of which were cloned in E. coli.

[0639] In one exemplary analysis, at least twenty-one recombinantnucleic acids (PRM15/trunE format) in a resulting library wereidentified as encoding recombinant polypeptides that reacted with denguevirus antibodies of at least three dengue virus serotypes by testing forantibody-antigen binding using the serotype specific anti-Denguevirus-types 1-4 antisera in FACS analyses performed under similarconditions as related experiments described in Examples 1-3. Thetwenty-one nucleic acid sequences were isolated, purified, and ligatedinto pMaxVax10.1 vectors, in accordance with the techniques applied inExample 1 (see, e.g., FIG. 2). The recombinant pMaxVax10.1 plasmids werecloned in E. coli to generate plasmids for the following DNAimmunization experiments.

[0640] A group of three mice was injected with 100 μg of plasmid DNA foreach one of the identified 21 pMaxVax10.1 plasmid vectors, each suchvector comprising a shuffled DNA sequence, or with the control vector,pMaxVax10.1_(null). Each mouse received a booster immunization of thesame dose of the same vector as the initial immunization at day 14, andwas subsequently bled on day 28 to obtain sera, according to the methoddescribed in Example 5. Sera obtained from the immunized mice wereanalyzed in a 1:100 dilution in PBS for antibody induction in ELISAassays on DEN-1, DEN-2, DEN-3, and DEN-4 inactivated virus coated ELISAplates, respectively, as described in Example 5. At least twelve of thenucleic acid sequences (PRM15/truncE format), 11B1 DNA (SEQ ID NO:173),11B8 DNA (SEQ ID NO:174), 11C11 DNA (SEQ ID NO:176), 11E2 DNA (SEQ IDNO:163), 12E3 DNA (SEQ ID NO:164), 12H4 DNA (SEQ ID NO:177), 13E2 DNA(SEQ ID NO:165), 13E11 DNA (SEQ ID NO:167), 13F11 DNA (SEQ ID NO:178),14B1 DNA (SEQ ID NO:179), 14E9 DNA (SEQ ID NO:166), and 14H2 DNA (SEQ IDNO:181), were identified by ELISA as encoding recombinant antigens, 11B1(SEQ ID NO:72), 11B8 (SEQ ID NO:73), 11C11 (SEQ ID NO:75), 11E2 (SEQ IDNO:76), 12E3 (SEQ ID NO:77), 12H4 (SEQ ID NO:78), 13E2 (SEQ ID NO:79),13E11 (SEQ ID NO:80), 13F11 (SEQ ID NO:81), 14B1 (SEQ ID NO:82), 14E9(SEQ ID NO:83), and 14H2 (SEQ ID NO:85), that induced in vivo productionof antibodies that reacted with dengue virus antigens of the four virusserotypes—Den-1, Den-2, Den-3, and Den-4—at levels well above thoseobserved with the control vector.

[0641] In a further round of experiments, mice were individuallyinjected with 100 μg of plasmid DNA of one of the following: (1) tenrepresentative plasmids (each comprising one of the 11B1, 11B8, 11C11,11E2, 12E3, 12H4, 13E2, 14B1, 14E9, and 14H2 nucleotide sequences), (2)plasmids comprising four parental nucleotide sequences described inExample 1 (i.e., pMaxVax10.1_(Den-1PRM15/tE CO),pMaxVax10.1_(Den-2PRM15/tE CO), pMaxVax10.1_(Den-3PRM15/tE CO), andpMaxVax10.1_(Den-4PRM15/tE CO)), (3) plasmids comprising four selectnucleic acids (2G11 DNA (SEQ ID NO:157), 6E12 DNA (SEQ ID NO:159), 5/21DNA (SEQ ID NO:158), and 2/7 DNA (SEQ ID NO:156)), or (4) thepMaxVax10.1_(null) plasmid vector. The immunization experiments wereperformed in triplicate with booster immunizations and bleedingperformed as described in Example 5. Sera obtained from the immunizedmice were analyzed for antibody induction in ELISA assays on DEN-1,DEN-2, DEN-3, and DEN-4 inactivated virus coated ELISA plates,respectively, as described in Example 5. Average OD values for seraobtained from groups of mice, each group comprised 3 mice, which hadbeen immunized with one type of plasmid against each type of ELISA platewere determined and graphically plotted. The results of thesecalculations are shown in FIG. 6. In this figure, the label “pMV” on theX axis of each plot refers to pMaxVax10.1_(null), and the labels “D-1,”“D-2,” “Den-3,” and “Den-4E” on the X axes refer topMaxVax10.1_(Den-1PRM15/tE CO), pMaxVax 10.1_(Den-2PRM15/tE CO), pMaxVax10.1 Den-3PRM 15/tE CO, and pMaxVax10.1_(Den-4PRM15/tE CO),respectively.

[0642] The results of these experiments, as shown in FIG. 6, demonstratethat all of the selected nucleic acids expressed antigens that inducedantibodies in mice that strongly reacted with all four inactivated WTdengue viruses (Den-1, Den-2, Den-3, and Den-4) in ELISA assays. Seraobtained from mice injected with plasmids comprising the selected secondround library nucleic acid sequences exhibited higher average OD valueson DEN-1, DEN-2, and DEN-3 ELISA plates than did the most relatedparental sequence; for example, sera from mice immunized with the secondround recombinant nucleic acids had higher average OD values on theDEN-1 plate than the average OD of mouse sera obtained from miceimmunized with pMaxVax10.1_(Den-1PRM15/tE CO) (labeled as D-1 in FIG.6). Furthermore, sera from mice that received injections of pMaxVax10.1plasmid DNA comprising at least 4 of the first second round nucleicacids—11C1-encoding DNA, 11B1 DNA, 11B8 DNA, and 14B1 DNA—as well seraobtained from mice that received injections of at leastpMaxVax10.1_(6E12) DNA, exhibited OD levels comparable to those levelsobserved with sera from mice that received injections ofpMaxVax10.1_(Den-4PRM15/tE CO) analyzed on DEN-4 specific ELISA plates.

[0643] The results of these experiments further demonstrate generationand identification of recombinant nucleic acids encoding recombinant(PRM15/tE) dengue antigens that induce or enhance production ofantibodies that react with (or bind or specifically bind to) multipledengue virus serotypes in vivo (as identified by the selecting screeningmethods described herein). The results of these experiments also confirmthat the nucleic acids and plasmid vectors of the invention (and theresulting recombinant polypeptides encoded therefrom) are capable of anduseful for inducing and enhancing such an immune response(s) in vivo insubjects, including mammals.

Example 7

[0644] This example illustrates the complex chimerism (i.e., sequencediversity) of select recombinant antigens of the invention where theamino acid sequences of such recombinant antigens are compared to aminoacid sequences of corresponding WT dengue virus antigens.

[0645] The antigens corresponding to the recombinant nucleotidesequences of the seven clones identified and selected as examples in thefirst round recombinant nucleotide library in Example 5 (i.e., 2/7 (alsotermed “6A11”), 5/21 (also termed “6D8”), 2G11, 6E12, 6C6, 6F4, and 7A9)and the antigens corresponding to the recombinant nucleotide sequencesof the 12 clones identified and selected in the first second roundrecombinant nucleotide library in Example 6 (11B1, 11B8, 11C11, 11E2,12E3, 12H4, 13E2, 13E11, 13F11, 14B1, 14E9, and 14H2) were sequenced andcompared with the polypeptide sequences of the corresponding sequenceregions (i.e., the PRM15 and truncated E protein (e.g., about 90% of theN terminus of the E protein)) of each wild-type dengue antigen for eachof the 4 dengue serotypes to determine regions of amino acid sequenceidentity between the recombinant and wild-type antigens using standardtechniques. Through such analysis, it was determined that therecombinant antigens included, e.g., various amino acid regions,fragments, or segments from the wild-type PRM15/truncated envelopeprotein polypeptide sequence for each of the 4 WT dengue virusserotypes. Approximate amino acid regions, fragments or segments in arecombinant antigen corresponding to a region, fragment or segment of awild-type antigen amino acid sequence were graphically plotted to assesschimerism of the recombinant antigens (data not shown) (see, however,the exemplary plot shown in FIG. 11). Additionally, it was noted thatdiversity increased with additional recursive sequence recombination,e.g., via DNA shuffling in combination with appropriate screening orselection procedures.

[0646] The above-described amino acid sequence analysis illustrates thecomplex chimeric nature (sequence diversity) of at least many of therecombinant antigens of the invention and nucleic acids encoding them.Moreover, the results of this sequence analysis establish that greaterdiversity (more complex chimerism) is induced in recombinant antigensencoded by DNAs produced and identified via multiple rounds of recursivesequence recombination combined with appropriate screening/selectionprocedures.

Example 8

[0647] This example describes the generation of a library of recombinantnucleic acids by recursive sequence recombination, the identification ofselect recombinant nucleic acids in such library using appropriatescreening/selection procedures, and the isolation of selected nucleicacids encoding recombinant dengue antigens from the library, which, whenexpressed in vivo, induced or enhanced the production of antibodies thatreacted with dengue viruses of multiple virus serotypes.

[0648] Representative recombinant dengue-antigen-encoding nucleic acidsequences from first round library clones 2G11, 6E12, 2/7 (6A11), and5/21 (6A8), described in Example 5, were isolated and purified usingstandard techniques and, in combination with the 4 parental denguesequences described in Example 1, used to produce a new library ofrecombinant nucleic acids by recursive sequence recombination inaccordance with the techniques described and/or referenced in Example 2and throughout the specification.

[0649] From this library of recombinant nucleic acids, twenty-fiverecombinant nucleotide sequences (in PRM15/tE format)) were selected viaflow cytometry (FACS) for immunization of mice and ELISA analyses of thesera using four serotype-specific inactivated dengue virus coated ELISAplates following the techniques described and referenced in Example 5.All 25 recombinant nucleotide sequences were analyzed as such and foundto induce or enhance production of cross-reactive antibodies against all4 DEN serotypes as discussed in detail below.

[0650] Specifically, twenty-five recombinant DNA sequences (in PRM15/tEformat)—(15C2 DNA (SEQ ID NO:182), 15D4 DNA (SEQ ID NO:183), 15H4 DNA(SEQ ID NO:184), 16B4 DNA (SEQ ID NO:185), 16E8 DNA (SEQ ID NO:168),16E10 DNA (SEQ ID NO:169), 16F12DNA (SEQ ID NO:186), 16G11 DNA (SEQ IDNO:187), 17A12 DNA (SEQ ID NO:188), 17D5 DNA (SEQ ID NO:189), 17D11 DNA(SEQ ID NO:190), 17F5 DNA (SEQ ID NO:191), 17F11 DNA (SEQ ID NO:192),17G5 DNA (SEQ ID NO:193), 17H3 DNA (SEQ ID NO:194), 17H10 DNA (SEQ IDNO:195), 17H12 DNA (SEQ ID NO:196), 18A9 DNA (SEQ ID NO:197), 18B7 DNA(SEQ ID NO:198), 18D7 DNA (SEQ ID NO:199), 18E9 DNA (SEQ ID NO:170),18E10 DNA (SEQ ID NO:171), 18E11 l DNA (SEQ ID NO:172), 18H2 DNA (SEQ IDNO:200), and 18H6 DNA (SEQ ID NO:235))—were isolated from the newlibrary of recombinant nucleic acids using techniques as described inExamples 1-7. A pMaxVax10.1 plasmid vector comprising each suchrecombinant DNA sequence was prepared as described above.

[0651] Mice were divided into groups of three. Three mice were eachimmunized by injection with one of the following DNA constructs: (1) 100μg of pMaxVax10.1 plasmid DNA vector comprising one of the 25recombinant sequences described above; (2) 100 μg of pMaxVax 10.1plasmid vector comprising the recombinant DNA sequence corresponding toclone 11C4 (SEQ ID NO:175) identified in a first second round library(see Example 5); (3) 100 μg of pMaxVax 10.1 plasmid vector comprisingthe recombinant DNA sequence corresponding to clone 14G10 DNA (SEQ IDNO:180) identified in a first second round library (see Example 5), and(4) 100 μg of pMaxVax10.1_(null) vector (control). Each mouse received abooster of the same dose of the same plasmid vector as the initialimmunization at days 14, 29, and 56 following the initial immunization(day 0). Sera were collected from the mice at days 28, 55, and 76. Thecollected sera were analyzed for in vivo antibody induction in ELISAassays on DEN-1, DEN-2, DEN-3, and DEN-4 inactivated virus coated ELISAplates in a 1:100 dilution under conditions described in Example 5.

[0652] The ELISA analyses of the mouse sera indicated that all 27plasmids (each comprising one of the PRM15/tE recombinant nucleotidesequences described above) lead to in vivo expression of antigens thatinduced production of antibodies that reacted with inactivated dengueviruses of all 4 virus serotypes in ELISA assays at both 28 and 55 daysafter injection. The average optical density (OD) value for eachrecombinant PRM15/tE antigen encoded by such recombinant plasmid vectorwas calculated for the sera obtained from each mice receiving suchplasmid injection(s) for each serotype-specific ELISA plate tested, ascompared to that value obtained using pMaxVax10.1_(null) (data notshown). The 25 recombinant PMR15/tE polypeptide antigens encoded by therecombinant nucleotide sequence identified in the new library includedthe following: (15C2 (SEQ ID NO:86), 15D4 (SEQ ID NO:87), 15H4 (SEQ IDNO:88), 16B4 (SEQ ID NO:89), 16E8 (SEQ ID NO:90), 16E10 (SEQ ID NO:91),16F12 (SEQ ID NO:92), 16G11 (SEQ ID NO:93), 17A12 (SEQ ID NO:94), 17D5(SEQ ID NO:95), 17D11 (SEQ ID NO:96), 17F5 (SEQ ID NO:97), 17F11 (SEQ IDNO:98), 17G5 (SEQ ID NO:99), 17H3 (SEQ ID NO:100), 17H10 (SEQ IDNO:101), 17H12 (SEQ ID NO:102), 18A9 (SEQ ID NO:103), 18B7 (SEQ IDNO:104), 18D7 (SEQ ID NO:105), 18E9 (SEQ ID NO:106), 18E10 (SEQ IDNO:107), 18E11 (SEQ ID NO:108), 18H2 (SEQ ID NO:109), and 18H6 (SEQ IDNO:110).

[0653] A further in vivo analysis was performed by injecting groups of 5mice each under identical conditions with 100 μg (50 μg/leg) of pMaxVax10.1_(null) or with 100 μg pMaxVax 10.1 DNA vector comprising one of thefollowing nucleotide sequences: 16B4 (SEQ ID NO:185), 16G11 (SEQ IDNO:187), 18H2 (SEQ ID NO:200), 18H6 (SEQ ID NO:235), Den-1PRM15/tE CO(SEQ ID NO:211), Den-2PRM15/tE CO (SEQ ID NO:212), Den-3PRM15/tE CO (SEQID NO:213), Den-4PRM15/tE CO (SEQ ID NO:214). Each mouse of anothergroup of 5 mice was injected with 100 μg (50 μg/leg) of a mixture ofpMaxVax10.1_(Den-1PRM15/tE CO), pMaxVax10.1_(Den-2PRM15/tE CO),pMaxVax10.1_(Den-3PRM15/tE CO), and pMaxVax10.1_(Den-4PRM15/tE CO) atthe same times and under the same conditions as described for the othergroups of mice that received individual injections of each of theabove-described plasmid DNA vectors. Each mouse received a boosterimmunization of the same dose of the same plasmid vector as the initialimmunization at days 14, 29, and 56 following the initial immunization(day 0). Sera were collected from the mice at days 28, 55, 76, 120 and180. Average OD values from sera at day 55 obtained for the experimentsare shown in FIG. 7.

[0654] The results of these further ELISA assays indicate that therecombinant PRM15/tE antigens (16B4 (SEQ ID NO:89), 16G11 (SEQ IDNO:93), 18H2 (SEQ ID NO:109), 18H6 (SEQ ID NO:110)) encoded by theselected recombinant nucleotides (16B4 (SEQ ID NO:185), 16G11 (SEQ IDNO:187), 18H2 (SEQ ID NO:200), 18H6 (SEQ ID NO:235)) induced productionof antibodies that reacted with dengue viruses of all 4 WT dengue virusserotypes. OD levels exhibited by antisera from mice injected with theseselected recombinant clones were significantly higher than thoseproduced by antigens (Den-1PRM15/tE (SEQ ID NO:149), Den-2PRM15/tE (SEQID NO:150), Den-3PRM15/tE (SEQ ID NO:151), Den-4PRM15/tE (SEQ IDNO:152)) encoded by any of the parental nucleotide sequences(Den-1PRM15/tE CO (SEQ ID NO:211), Den-2PRM15/tE CO (SEQ ID NO:212),Den-3PRM15/tE CO (SEQ ID NO:213), Den-4PRM15/tE CO (SEQ ID NO:214)),individually or in combination, for the DEN-1, DEN-2, and DEN-3 viruscoated ELISA plates. Antisera from mice injected with plasmidscomprising these recombinant antigen-encoding sequences also had ODlevels at least as high as any of these parental antigen-encodingsequences against DEN-4 (individually or in combination) (high OD levelsexhibited by sera obtained from pMaxVax10.1_(Den-3PRM15/tE CO)-injectedmice on such plate may suggest a flaw in the ELISA plate used for thisportion of the experiment).

[0655] The results of these experiments further demonstrate the abilityof the recombinant antigens of the invention, and/or recombinant nucleicacids of the invention that encode recombinant antigens, to induce orenhance production of antibodies that react with (or bind orspecifically bind to) dengue viruses of all four serotypes in vivo. Suchrecombinant antigenic polypeptide sequences of the invention, and suchrecombinant nucleotide sequences encoding recombinant antigenicpolypeptides of the invention, are useful, e.g., in prophylactic and/ortherapeutic methods of the invention for the induction, modulation,and/or enhancement of the production of antibodies that react with (orbind or specifically bind to) dengue viruses of all four serotypesand/or diagnostic assays to detect the presence of antibodies in abiological sample to 1, 2, 3, and 4 dengue virus serotypes.

Example 9

[0656] This example describes the ability of recombinant antigens of theinvention to produce, enhance, modulate, and/or promote a neutralizingantibody response(s) against dengue viruses of multiple dengue virusserotypes in vivo.

[0657] Mice were individually injected with a pMaxVax10.1 plasmid vectorcomprising a recombinant DNA sequence corresponding to one of thefollowing—18E9 (SEQ ID NO:170), 18D7 (SEQ ID NO:199), 16G11 (SEQ IDNO:187), 18H6 (SEQ ID NO:235), 15D4 (SEQ ID NO:183), 18H2 (SEQ IDNO:200), 6E12 (SEQ ID NO:159), 2/7 (SEQ ID NO:156), 2G11 (SEQ IDNO:157), and 16B4 (SEQ ID NO:185)—according to the methods set forth inExample 8. These DNA sequences encoded the following recombinantPRM15/tE antigens, respectively: 18E9 (SEQ ID NO:106),), 18D7 (SEQ IDNO:105), 16G11 (SEQ ID NO:93), 18H6 (SEQ ID NO:110), 15D4 (SEQ IDNO:87), 18H2 (SEQ ID NO:109), 6E12 (SEQ ID NO:69), 2/7 (SEQ ID NO:65),2G11 (SEQ ID NO:66) and 16B4 (SEQ ID NO:89). In a similar experimentDEN-1, DEN 2, DEN-3, and DEN-4 wild type sequences, coding for theDEN-1-4 wild-type PRM15/tE antigens, and a equal mix of these 4wild-type antigens were injected. All mice received 3 booster injectionswith the same plasmid DNA in 2-week intervals.

[0658] Mice were also individually injected with a pMaxVax10.1 plasmidvector comprising a recombinant DNA sequence corresponding to one of thefollowing—5/21-D1 (SEQ ID NO:201), 2G11-D4 (SEQ ID NO:204), and 6E12-D4(SEQ ID NO:202). These DNA sequences encoded the following recombinantfull length C15/full prM/full length E antigens, respectively:following—5/21-D1 (SEQ ID NO:140), 2G11-D4 (SEQ ID NO:139), and 6E12-D4(SEQ ID NO:141). In a similar experiment DEN-1, DEN-2, DEN-3, and DEN-4wild type sequences, coding for the wild-type C15/full prM/full length Eantigens, and a equal mix of these 4 wild-type antigens were injected.All mice received 3 booster injections with the same plasmid DNA in2-week intervals.

[0659] Antisera obtained from the mice 76 days after initial DNAinjection were analyzed by standard plaque reduction neutralizationtiter (PRNT) assay, which is well known to those of ordinary skill inthe art (see, e.g., Russell et al., J Immunol (1967) 99:285-290; Simmonset al, Am. J. Trop. Med. Hyg (2001) 65:420-426), each incorporatedherein by reference in its entirety for all purposes.

[0660] Briefly, this PRNT₅₀ assay is typically conducted as follows:Cell cultures of monkey kidney cells (LLC-MK2) are seeded in 6 wellculture plates and incubated at 37° C. in a CO₂ incubator. Each of theantisera obtained from the mice injected with one of the 9 plasmidvectors is diluted to make 1:20, 1:40, and 1:80 serial dilutions. Eachcell culture is incubated with a mixture of (i) dengue viruses of eachof the four 4 dengue virus serotypes and (ii) a 1:20, 1:40, or 1:80serial dilution of antisera for 2 to 3 hours.

[0661] After incubation of 2-3 hours, the inoculum mixture of dengueviruses and diluted antisera are removed from the LLC-MK2 monkey kidneycell cultures, and a layer of agarose (SeaPlaque agarose, FMCBioproducts) is added to the cell cultures. Plaques formed by thereleased virus progeny are visualized at day 7 by staining with a 0.02%neutral red/Hanks balanced salt solution. The plaque counts for eachsuch cell culture are compared to plaque counts for LLC-MK2 cellsincubated with an identical mixture of dengue viruses without anyantisera. The specific determination of 50% plaque reductionneutralization titers (PRNT₅₀) for the cultures is facilitated by use ofProbit analysis software (SSPS, Inc. Chicago, Ill.), using standardtechniques and according to manufacturer's instructions. The 50%effective dose (ED₅₀) is the serum dilution that caused a 50% reductionin the number of plaques. The amount of antiserum (e.g., serum from asubject containing specific antibodies produced by immunization with aspecific immunogen) required to neutralize 50% of the infectious virusparticles included in a specific virus challenge dose is directlyrelated to the potency of the antiserum. Russell et al., supra, at 286.

[0662]FIG. 8A shows the results of PRNT₅₀ analyses for these 10recombinant PRM15/tE antigen-encoding DNA sequences (e.g., clones 18E9,18D7, 16G11, 18H6, 15D4, 18H2, 6E12, 2/7, 2G11, and 16B4), the four DENwild-type PRM15/tE antigens (DEN-1, DEN-2, DEN-3, and DEN-4)individually, and an equal mix of these four DEN wild-types. FIG. 8Bshows the results of PRNT₅₀ analyses for these 3 recombinant full lengthC15/full prM/full length E antigen-encoding DNA sequences (e.g., clones5/21-D1, 2G11-D4, and 6E12-D4), the four DEN wild-type C15/full prM/fulllength E antigens (DEN-1, DEN-2, DEN-3, and DEN-4) individually, and anequal mix of these four DEN wild-types. Reciprocal PRNT₅₀ titers of >20were regarded as positive for production of neutralizing antibodiesagainst a particular dengue virus serotype used for the in vitroneutralization assay. Applying this standard to the PRNT₅₀ titerspresented in FIGS. 8A and 8B, all 13 of these recombinant plasmidvectors induced production of neutralizing antibodies against at least 2dengue viruses (e.g., against at least two of DEN-1, DEN-2, DEN-3,and/or DEN-4). For example, injection of mice with pMaxVax10.1_(18E9),induced neutralizing antibodies against at least DEN-1 and DEN-2.Injection of mice with pMaxVax10.1_(18D7), pMaxVax10.1_(15D4), andpMaxVax10.1_(6E12) induced neutralizing antibodies against at leastDEN-1, DEN-2, and DEN-3. Injection of mice with pMaxVax10.1_(2/7),pMaxVax 10.1_(2G11),pMaxVax 10.1_(16G11), pMaxVax10.1_(18H6),pMaxVax10.1_(18H2), and pMaxVax10.1_(16B4), pMaxVax10.1_(5/21-D1),pMaxVax10.1_(2G11-D4), and pMaxVax 10.1_(6E12-D4) induced production ofneutralizing antibodies against all four dengue virus serotypes, DEN-1,DEN-2, DEN-3, and DEN-4. In contrast, only a mix of the wild-type DEN-1,DEN-2, DEN-3, and DEN-4, coding for the recombinant full length C15/fullprM/full length E antigens, induced production of neutralizingantibodies against all four dengue virus serotypes, DEN-1, DEN-2, DEN-3,and DEN-4. The two DEN wild-type PRM15/tE antigens DEN-1, and DEN-2, aswell as a mix of the four DEN wild-type PRM15/tE antigens (DEN-1, andDEN-2, DEN-3, and DEN-4), and the two DEN-wild-type full length C15/fullprM/full length E DEN-1, and DEN-2 antigens, did not induce neutralizingantibodies. The two DEN wild-type PRM15/tE antigens DEN-3, and DEN-4induced only neutralizing antibodies against the homologous DEN-virus(DEN-1 and DEN-2, respectively), while the two DEN wild-type full lengthC15/full prM/full length E antigens, DEN-3, and DEN-4, inducedneutralizing antibodies against at least 3 Den viruses (DEN-2, DEN-3,and DEN-4).

[0663] ELISA analyses were also performed with sera obtained from miceinjected with 100 μg pMaxVax10.1 plasmid vector comprising a recombinantnucleotide sequence corresponding of one of each of these 13 clones(clones 18E9, 18D7, 16G11, 18H6, 15D4, 18H2, 6E12, 2/7, 2G11, 16B4,5/21-D1, 2G11-D4, and 6E12-D4) and sera obtained from mice injected withpMaxVax10.1_(null) using DEN-1, DEN-2, DEN-3, and DEN-4 virus coatedELISA plates and DNA immunization techniques according to the methodsdescribed in Example 8. The resulting ELISA data demonstrated that all13 of these recombinant clones induced production of antibodies in vivothat reacted with all four dengue virus serotypes in vitro (data notshown).

[0664] The results of these experiments demonstrate that the recombinantnucleotide sequences encoding recombinant dengue antigens (for thePRM15/tE format and the C15/full prM/full length E format) of theinvention and/or recombinant dengue antigens ((for the PRM15/tE formatand the C15/full prM/full length E format)) of the invention induced,enhanced, promoted, and/or modulated production of neutralizingantibodies to at least 2, 3, or even 4 dengue virus serotypes when suchrecombinant antigenic polypeptides are expressed in vivo. Suchrecombinant antigenic polypeptides of the invention, and suchrecombinant nucleotide sequences encoding recombinant antigenicpolypeptides of the invention, are useful, e.g., in prophylactic/and ortherapeutic methods of the invention for the induction, modulation,and/or enhancement of the production of neutralizing antibodies to atleast 2, 3, or even 4 dengue virus serotypes when such recombinantantigenic polypeptides are expressed in vivo.

Example 10

[0665] This example illustrates the secretion characteristics ofrecombinant dengue antigens determined to induce, enhance, promote,and/or modulate neutralizing antibody production against dengue virusesof at least 2 serotypes in vivo.

[0666] The plasmid DNA corresponding to the pMaxVax10.1 vectorcomprising the recombinant nucleotide sequence of each of clones 18D7,18E11, 16G11, 18H6, 18H2, 16B4, 14G10, 18E9, and 18E10 was isolated andpurified by standard techniques and used to transfect 293 cells asdescribed above and in Example 1 and 2. The transfected cells werecultured for 72 hours, and 15 μl of the unconcentrated cell-free mediumsupernatants subjected to polyacrylamide gel electrophoresis andmembrane blotting. The nitrocellulose membranes were incubated with theanti-DEN-1, DEN-3, and DEN-4 antibodies from mouse ascitic fluid andappropriate enzyme-conjugated secondary antibodies to produce a Westernblot using the techniques described above and in Example 1. FIG. 9 is avisualization of the Western blot obtained by this technique; the number(N) of wild-type dengue virus serotypes neutralized by the respectiverecombinant clones, as discussed in Example 9, is indicated on thebottom of the Western blot.

[0667] As shown in FIG. 9, 5 of the recombinant clones (18E9, 18E10,18E11, 18H12, and 18H6) expressed and secreted recombinant antigens thatproduced dark bands on the Western blot, whereas three recombinantantigens (14G10, 16B4, 18D7) were well secreted, but not strongly boundby the antibodies, resulting in weaker bands. One recombinant clone(16G11) was not secreted at a detectable level in the unconcentratedsupernatants. A comparison of the number of WT dengue virus serotypesneutralized by each recombinant antigen with its secretion profileindicated that there was no direct relationship between recombinantantigen secretion and the number of WT dengue virus serotypesneutralized by antibodies induced by the respective recombinantantigens. For example, the recombinant antigen that was not detectablysecreted (16G11) produced neutralizing antibodies against all four WTdengue virus serotypes. The well-expressed and secreted recombinantantigens that produced dark bands (18E9, 18E10, 18E11, 18H12, and 18H6)produced neutralizing antibodies against two, two, four, four, and fourWT dengue virus serotypes, respectively. The recombinant antigens thatproduced weaker bands (14G10, 16B4, 18D7) produced antibodies thatneutralized two, four, and three 2 WT dengue virus serotypes,respectively.

[0668] The results of these experiments demonstrate that both secretedand cell membrane bound recombinant antigens of the invention (and therecombinant nucleotide sequences encoding such recombinant antigens) areeffective in inducing, enhancing, promoting, and/or modulatingproduction of neutralizing antibodies against one or more WT denguevirus serotypes in vivo.

Example 11

[0669] This example illustrates the longevity of the antibody responseto recombinant dengue virus antigens induced by in vivo injection of DNAplasmids comprising recombinant nucleotide sequences of the inventionthat encode recombinant antigens of the invention.

[0670] An immune response against an infectious agent (e.g., protectiveimmune response) preferably is one that is long-lasting. To test thelongevity of the antibody immune response in a subject (e.g., mammal)receiving one or more injections of DNA plasmids encoding recombinantdengue antigens of the invention that induce neutralizing Ab responsesagainst at least 2 dengue virus serotypes, the following experimentswere performed.

[0671] Three mice each were injected with a pMaxVax10.1 DNA plasmidvector, wherein each such vector encoded a recombinant antigencorresponding to one of each of the following eight recombinantclones—16B4 (SEQ ID NO:89), 16G11 (SEQ ID NO:93), 18D7 (SEQ ID NO:105),18E9 (SEQ ID NO:106), 18E10 (SEQ ID NO:107), 18E11 (SEQ ID NO:108), 18H2(SEQ ID NO:109), or 18H6 (SEQ ID NO:110), at set time intervals.(Alternatively, mice each were injected with a pMaxVax10.1 DNA plasmidvector, wherein each such vector comprising a nucleic acid correspondingto one of each of the following eight recombinant clones 16B4 (SEQ IDNO:185), 16G11 (SEQ ID NO:187), 18D7 (SEQ ID NO:199), 18E9 (SEQ IDNO:170), 18E10 (SEQ ID NO:171), 18E11 (SEQ ID NO:172), 18H2 (SEQ IDNO:200), or 18H6 (SEQ ID NO:235). Antisera were obtained from these miceusing techniques described above (e.g., Example 5). Sera collected fromthe mice at 55 and 120 days, respectively, after initial DNAimmunization (day 0) by injection with these recombinant pMaxVax10.1plasmid vectors were subjected to ELISA analyses, as described above,using, e.g., inactivated DEN-2 virus coated ELISA plates (all of theserecombinant clones had previously been shown to induce a tetravalentantibody response in similar DNA plasmid injection experiments in vivo).The average OD values for antisera of mice injected with each type ofplasmid were calculated at both test periods. The antibody responseinduced by each recombinant clone tested remained high at 55 days afterthe initial injection of the corresponding recombinant pMaxVax10.1plasmid vector (comprising the recombinant nucleic acid encoding therecombinant antigen). Remarkably, the antibody responses induced byinjection of these recombinant pMaxVax 10.1 vectors were substantiallyunchanged at 120 days after the initial pMaxVax10.1 vector injection ascompared to 55 days after the initial pMaxVax10.1 vector injection. Forexample, antibody responses induced by injection of these recombinantvectors were increased or decreased within a range of from about 0.5%,about 1%, about 2%, about 5%, about 7%, about 10%, about 12%, or about14%, at 120 days after the initial injection as compared to 55 daysafter the initial injection.

[0672] The results of these experiments demonstrate that recombinantantigens of the invention, and recombinant nucleic acids encoding suchantigens, are capable of inducing and/or promoting an in vivo antibodyresponse against multiple WT dengue virus serotypes over sustainedperiods of time, including e.g., over at least about 55 and 120 days.

[0673] Such antibody response(s) may be induced and/or promoted, e.g.,by: (1) in vivo or ex vivo administration to a subject of a recombinantDNA plasmid vector comprising a nucleotide sequence that encodes arecombinant antigen of the invention (or a recombinant DNA plasmidvector comprising a recombinant nucleotide sequence of the invention) inan amount sufficient or effective to induce or promote such desiredantibody response(s); or (2) by in vivo or ex vivo administration to asubject or cells of the subject of a recombinant antigen (or nucleicacid encoding such antigen) of the invention, or chimeric virus or VLPof the invention, in an amount effective to induce or promote suchdesired antibody response(s). Such antibody responses are also observedin in vitro or ex vivo assays using antisera obtained from suchsubjects. The desired Ab response may be, e.g., an antibody responsethat is sufficient for prophylactic and/or therapeutic treatment of adisease or disorder (including as, e.g., a prophylactic agent or vaccineagainst dengue infection or dengue fever). Administration of suchrecombinant DNA plasmid or recombinant antigenic polypeptide to asubject may be according to any in vivo or ex vivo method for deliveryor administration of a nucleic acid or polypeptide (or pharmaceuticalcomposition thereof) to a subject as described herein and throughoutthis specification, including, but not limited to, e.g., injection orgene gun delivery, and including dosages and/or compositions describedherein, which may be dependent upon the particular application ortreatment method of interest.

Example 12

[0674] This example illustrates the production of recombinant denguevirus antigens, and recombinant nucleic acids encoding recombinantdengue antigens, that induce the production of neutralizing antibodiesagainst multiple dengue virus serotypes in vivo, wherein each such arecombinant dengue virus antigen comprises an amino acid sequence havinga length (in amino acid residues) identical, substantially identical(e.g., having at least about 75%, 80%, 85%, 86%, 87%, 88% or 89%,preferably at least about 90%, 91%, 92%, 93%, or 94%, and morepreferably at least about 95% (e.g., about 87-95%), 96% 97%, 98%, 99%,99.5% identity in length), equivalent, or substantially equivalent to(e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% ormore identical in length to) the length of a fusion protein comprisingor consisting of the full length amino acid sequence of a prM proteinfused to the full length amino acid sequence of an envelope (E) proteinof a dengue virus of a particular serotype. In another aspect, each suchrecombinant dengue virus antigen is encoded by a nucleotide sequencehaving a length equivalent or substantially equivalent to (e.g., atleast about 95%, about 96%, about 97%, about 98%, about 99% or moreidentical in length to) the length of a nucleotide sequence encoding afusion protein comprising or consisting of the full length prM proteinsequence fused to the full length E protein sequence of a specificdengue virus serotype.

[0675] As described above, for selected methods of recursive sequencerecombination, the following codon optimized dengue nucleotide sequenceswere used as parental sequences: Den-1 PRM15/tE CO, Den-2 PRM15/tE CO,Den-3 PRM15/tE CO, or Den-4 PRM15/tE CO. For each dengue virus serotype,each such parental nucleotide sequence comprised a codon optimizednucleotide sequence encoding a wild-type dengue fusion proteincomprising: 1) a “PRM15 polypeptide” (e.g., a polypeptide sequencecomprising an initial methionine (Met) residue and the last 15 aminoacids of the C terminus of the prM protein of a WT dengue virus of aspecific serotype, e.g., DEN-1); and 2) a truncated E protein, whereinthe truncated E protein comprised from at least about 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, or 95% or more of the contiguous aminoacid residues of the full length E protein sequence of the same (WT)dengue virus serotype, as measured or beginning from about the Nterminal amino acid residue of the E protein sequence. That is, thetruncated E protein comprised a sequence of contiguous amino acidresidues of at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,or 95% or more of the contiguous amino acid residues of the full lengthE protein sequence of the same (WT) dengue virus serotype, as measuredfrom about the N terminal amino acid residue of the E protein sequence.

[0676] For each wild-type dengue virus of one of the four serotypes, acodon optimized nucleotide sequence encoding a fusion protein comprisingthe full length prM protein and full length E protein of that serotypewere made. In one aspect, each such codon optimized nucleotide sequencewas made via nucleotide extension of the truncated parental Den-1PRM15/tE CO, Den-2 PRM15/tE CO, Den-3 PRM15/tE CO, or Den-4 PRM15/tE COnucleotide sequence, respectively. To generate a nucleotide sequenceencoding a fusion protein comprising a full length prM protein fused toa full length E protein (or, e.g., to generate a nucleotide sequencecomprising a full length prM nucleotide sequence/full length Enucleotide sequence) via nucleotide extension for each of the codonoptimized truncated parental nucleotide sequences (e.g., Den-1 PRM15/tECO, Den-2 PRM15/tE CO, Den-3 PRM15/tE CO, and Den-4 PRM15/tE CO), codonoptimized nucleotide sequences encoding the full length prM proteinsequence and full length E protein sequence (i.e., the amino acidresidues of the C terminus of E protein needed to be added to thecorresponding truncated E protein of the particular dengue virusserotype to make a full length E protein) were determined for eachdengue virus serotype (Den-1, Den-2, Den-3, and Den-4) in accordancewith the methods described in Example 1. Each codon optimized truncatedparental nucleotide sequence (e.g., Den-1 PRM15/tE CO, Den-2 PRM15/tECO, Den-3 PRM15/tE CO, or Den-4 PRM15/tE CO nucleotide sequence) wasanalyzed for unique restriction site(s) at the 5′ and 3′ ends of thenucleic acid sequence of the E gene.

[0677] By performing such an analysis, it was determined that the codonoptimized E genes for all 4 dengue virus serotypes included a uniqueBsrBI restriction site at about position 57 in the E gene for eachparticular virus. The BsrBI restriction site also was found in therespective recombinant dengue antigen-encoding nucleotide sequences ofclones 5/21, 2/7, 2G11, and 6E12, facilitating 5′ extension of thesenucleotide sequences with additional nucleic acid residues necessary tomake a corresponding full length (complete) prM gene sequence of aparticular serotype, as further described below.

[0678] For each dengue virus of the 4 dengue virus serotypes, codonoptimized nucleotide sequences also were generated for the full lengthprM gene and the nucleotide sequence encoding a 16-amino acid sequencecomprising Met as the first amino acid residue and the (last) 15 aminoacid residues from the C terminus of the capsid (C) gene of theparticular dengue virus serotype. Such 16-amino acid sequence served asa signal sequence. Codon optimized prM genes of the dengue viruses ofthe 4 serotypes, including the nucleotide sequence encoding such 16amino acid signal sequence, and the 5′ sequences of the E geneoverlapping the BsrB1 site, were synthesized by oligonucleotideassembly, as described in Example 1.

[0679] Sequence analysis also was able to identify unique restrictionsites positioned near the 3′ end of the various dengue E genes.Specifically, a unique BsaBI site was identified at position 1793 in thecodon optimized DEN-1 prM/E gene sequence; a unique SexAI site wasidentified at position 1793 in the codon optimized DEN-2 prM/E genesequence; a unique BglII site was identified at position 1863 in thecodon optimized DEN-3 prM/E gene sequence; and a unique BsrGI site wasidentified at position 1884 in the codon optimized DEN-4 prM/E genesequence.

[0680] For example, to extend the nucleic acid sequence of Den-4PRM15/tE CO to a length equivalent or substantially equivalent to thelength of a full length C15/full prM/full length E protein fusionprotein, a human codon optimized nucleotide sequence corresponding tothe 3′ end of the DEN-4 E gene encoding the “C terminal extension”(e.g., a CO sequence comprising the nucleotide residues needed to beadded to a truncated E gene to extend the truncated E gene to make afull length E gene) was also synthesized by standard nucleotidesynthesis techniques. The synthesized codon optimized Den-4 C15/prMfragment (comprising a sequence of 16 amino acids—15 amino acids of theC terminal of the C protein and the first Met—and the additional Nterminal nucleotide residues needed to extend the codon PRM15 nucleotidesequence of a particular serotype to make a full length CO C15/full prMnucleotide sequence) was digested with BsrBI, the synthesized 3′ end ofDEN-4 E gene (encoding such codon optimized C terminal extensionnucleotide sequence) was digested with BsrG1, and the Den-4 PRM15/tE COnucleotide sequence was digested with BsrBI and BsrG1 according tomanufacturer's instructions. The three resulting fragments were ligatedby standard techniques to extend the PRM15 and truncated E genesequences to form a codon optimized Den-4 full length C15/full prM/fulllength E nucleotide sequence, designated “Den-4 C15/prM/E.” The samemethods were used to synthesize codon optimized C15 signal sequence/fulllength prM/full length E nucleotide sequences corresponding to each ofDen-1, Den-2, and Den-3, respectively, using the specific uniquerestriction sites discussed above. The term “C15 signal sequence”typically refers to a 16 amino acid sequence that includes as the firstcodon a codon encoding a Met residue and subsequent codons that encodethe 15 amino acids at the C terminus of the capsid protein of a denguevirus of one of the four serotypes. The resulting “full-length” codonoptimized C15/full prM/full E nucleotide sequences code for each of theparental Den-1 C15/full prM/full E, Den-2 C15/full prM/full E, and Den-3C15/full prM/full E polypeptides and were termed Den-1 C15/full prM/fullE, Den-2 C15/full prM/full E, Den-3 C15/full prM/full E nucleotides,respectively. In this instance, the term “C15 signal sequence” refers toa 16 amino acid sequence that includes as the first codon a Met residueand subsequent codons that encode the 15 amino acids of the C terminusof the capsid protein, “prM” or “full prM” refers to a full length prMpolypeptide or nucleic acid sequence, and the term “E” or “full E”refers to a full length E protein or nucleic acid sequence, dependingupon context.

[0681] Using a parental Den-1 C15/full prM/full E codon optimizednucleotide sequence, digestions were performed with appropriaterestriction enzymes (essentially as described above for the parentalgenes) to extend the nucleotide sequence of clone 2/7 (PRM15/tE format;SEQ ID NO:156) with a codon optimized nucleotide sequence encoding a 16amino acid sequence comprising a Met residue as the first amino acidlinked to the last 15 contiguous amino acids of the C terminal of theDEN-1 C protein, a portion of the N-terminal portion of DEN-1 prMprotein, and codon optimized nucleotide sequence fragments encoding aportion of the C-terminus of DEN-1 E protein, sufficient to generate,upon appropriate ligation the 2/7 nucleotide sequence, a recombinantnucleotide sequence comprising a C15/full length prM/full length Eformat nucleic acid sequence, termed extended “2/7-D1” (SEQ ID NO:203).Recombinant “extended” codon optimized nucleotide acid 2/7 (SEQ IDNO:203) encoded recombinant 2/7-D1 (C15/full prM/full E format)polypeptide (SEQ ID NO:147).

[0682] The nucleic acid sequence of clone 5/21 (PRM15/tE format; SEQ IDNO:158) was similarly extended using nucleotide sequence fragments ofthe DEN-1 C protein, DEN-1 prM protein, and DEN-1 E protein to generatea recombinant extended codon optimized nucleotide sequence 5/21-D1(extending clone 5/21 in PRM15/tEpolypeptide-encoding polynucleotideformat to 5/21-D1 in C15/full prM/full E polypeptide-encodingpolynucleotide format using wild-type Den-1 C terminal E proteinfragment-encoding nucleic acid and wild-type Den-1 N terminalC15/truncated prM polypeptide-encoding nucleic acid for extension) (SEQID NO:201), which encoded recombinant 5/21-D1 (C15/full prM/full Eformat) polypeptide (SEQ ID NO:201).

[0683] Similarly, the parental DEN-4 C15/full prM/full E codon optimizednucleotide sequence was used to extend the nucleotide sequences of 2G11(SEQ ID NO:157) and 6E12 (SEQ ID NO:159) to create “extended” 2G11-D4and 6E12-D4 nucleotide sequences in C15/full length prM/full length Enucleotide sequence format (SEQ ID NOS:204 and 202, respectively), whichencoded recombinant 2G11-D4 and 6E12-D4 (C15/prM/E format) polypeptides(SEQ ID NOS:139 and 141, respectively). All such resulting recombinantnucleic acids in C15 nucleic acid/full length prM nucleic acid/fulllength E nucleic acid sequence format were amplified and cloned intopMaxVax10.1 vectors, as described above. The C15 nucleic acid/fulllength prM nucleic acid/full length E nucleic acid sequence format isconveniently referred to as the “C15/prM/E” or “C15/full prM/full E”format.

[0684] Mice were injected with pMaxVax10.1_(6E12-D4) andpMaxVax10.1_(5/21-D1) plasmid vectors and the antiserum therefromcollected at 76 days after initial DNA plasmid vector injection, usingthe techniques described in Example 9. Non-extended clonespMaxVax10.1_(6E12) and pMaxVax10.1_(2G11) also were injected into miceunder similar conditions. Antisera from these mice also were collectedat 76 days after initial DNA plasmid vector injection. The antiseraobtained from these mice were used in a plaque reduction neutralizationtiter assay as described in Example 9, and the inverse PRNT₅₀ titersfrom these assays were calculated and compared to the results of theexperiments described in Example 9. The combined reciprocal PRNT₅₀titers observed in the two sets of experiments are set forth in FIGS. 8Aand 8B.

[0685] As shown in FIGS. 8A and 8B, both of the extended nucleotidesequences corresponding to “extended” clones 5/21-D1 and 6E12-D4(C15/full prM/full E format) produced according to this techniqueencoded recombinant antigens that induced neutralizing antibodiesagainst all 4 dengue serotypes in vivo, based on the PRNT assaystandards (>20; dilution of serum (reciprocal)) provided in Example 9.In contrast to the nucleotide sequence of 6E12-D4 (C15 nucleic acid/fulllength prM nucleic acid/full length E nucleic acid format), whichproduced inverse (reciprocal) PRNT₅₀ titers of above 80 for eachserotype, the nucleotide sequence of clone 6E12 (PRM15/truncated E geneformat) produced reciprocal PRNT₅₀ titers of 35, >80, 70, and 20 forDen-1, Den-2, Den-3, and Den-4, respectively (i.e., except for Den-2,lower inverse PRNT₅₀ scores were observed for Den-1, Den-3, and Den-4for clone 6E12 as compared to clone 6E12-D4). The nucleotide sequencecorresponding to clone 2G11 also induced neutralizing antibodies againstall 4 dengue virus serotypes.

[0686] These experiments demonstrate a method for producing recombinantdengue antigen-encoding nucleotide sequences, each of which encodes afusion protein that is identical or substantially identical in length(e.g., at least about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably atleast about 90%, 91%, 92%, 93%, or 94%, and more preferably at leastabout 95% (e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% identity inlength) to a wild-type dengue virus “C15/full length prM protein/fulllength E protein” fusion protein. Such recombinant nucleotide sequencesencode recombinant “C15/full length prM protein/full length E” fusionproteins. Plaque reduction neutralization assay analysis of suchrecombinant fusion protein demonstrated that extension of thepolypeptide sequence of a recombinant PRM15/trunE dengue antigen withappropriate wild-type sequence fragments to produce an “extended”C15/full prM/full E dengue antigen may increase the number of denguevirus serotypes the antibodies induced by such extended recombinantantigens neutralize in vivo and the reciprocal PRNT titer of suchneutralizing antibody responses relative to recombinant PRM15/truncEdengue antigens.

[0687] It was previously shown for tick-borne encephalitis (TBE),another flavivirus, that expression of the viral prM gene and 100% ofthe E gene of TBE can lead to viral polypeptide(s) forming a viral-like(empty) particle (VLP), which can have physical and antigeniccharacteristics that are substantially similar or identical to those ofa whole virus. A VLP is not an infectious particle because it doestypically not contain viral genetic material capable of producing liveviruses.

[0688] The present invention also includes dengue viral-like particles(VLPs), each of which comprises a dengue polypeptide sequence of theinvention, including, e.g., but not limited to: (1) a fusion proteincomprising a recombinant C15 signal peptide/full length prM protein/fulllength E protein; (2) a fusion protein comprising a recombinant fulllength prM protein/full length E protein (with or without any signalsequence, including any flavivirus signal peptide, described herein);(3) a fusion protein comprising a recombinant full length M protein/fulllength E protein (with or without any pr segment or fragment of a prsegment, described herein) and (4) a recombinant full length E protein(with or without any signal sequence, including any prM or fragmentthereof, described herein);

[0689] In the present case, recombinant VLPs of the invention can bemade, e.g., as follows. Cells (e.g., 293 cells) are transfected with aplasmid vector comprising a recombinant nucleotide sequence encoding arecombinant protein or fusion protein of the invention (e.g., includingany of those described in (1) to (4) above. The transfected DNA sequenceis translated into the corresponding recombinant protein or fusionprotein, respectively; in some instances, where a recombinant fusionprotein is produced, such fusion protein which may be subsequentlycleaved by a protease in the cell into its components, yielding, e.g., afull length prM protein and full length E protein; C15 signal peptide; Mprotein and E protein; and pr segment (typically degraded). One suchexpressed protein/peptide associates or assembles with at least oneother such protein/peptide, forming oligomers and such oligomersassemble to form recombinant VLPs in the cells. The mature particles arereleased from the cells into the medium by exocytosis. In someembodiments, the resulting VLPs may further comprise, associate with, orassemble with cellular membrane material. In some instances, the signalpeptide sequence is not included in the resulting recombinant VLP.Following expression and formation, the VLPs of the invention can beisolated by, e.g., gradient centrifugation or other methods known in theart. Such recombinant VLPs of the invention are useful in methods forthe prophylactic and/or therapeutic treatment of diseases or disordersdescribed herein or in diagnostic assays described herein forsimultaneous detection or diagnosis of antibodies against two or more(e.g., two, three, four) serotypes of dengue virus in a sample, such asa biological sample from a subject, such as a human patient at risk fordengue virus infection.

Example 13

[0690] This example further demonstrates the improved expression and/orsecretion of recombinant dengue virus antigens of the invention.

[0691] 293 cell cultures were prepared and transfected withpMaxVax10.1_(2G11), pMaxVax10.1_(18H6), pMaxVax10.1_(Den-3PRM15/tE CO),pMaxVax10.1_(Den-4PRM15/tE CO), and pMaxVax10.1_(null) plasmid vectors.The transfected cells were incubated for 72 hours under conditionspermissive for transgene expression (or heterologous nucleotideexpression) and secretion. A control vector that did not include adengue virus nucleotide sequence (pMaxVax10.1_(null) vector) wasincluded for comparison. The cell-free medium from each such culture wasobtained, subjected to standard polyacrylamide electrophoresis andmembrane blotting, and the membrane incubated with a mix of anti-DEN-1,DEN-3, and DEN-4 antibodies from mouse ascitic fluid and appropriateenzyme-conjugated secondary antibodies in accordance with the techniquesdescribed in Example 1 to produce the Western blot. The lanescorresponding to recombinant 2G11 and 18H6 dengue antigens, expressedfrom pMaxVax10.1_(2G11) and pMaxVax10.1_(18H6), respectively, containedsignificantly more dengue E protein, as reflected in darker, broaderbands, than was observed in the lanes corresponding to wild-typeDen-3PRM15/tE and Den-4PRM15/tE dengue antigens, expressed frompMaxVax10.1_(Den-3PRM15/tE CO) and pMaxVax10.1_(Den-4PRM15/tE CO),respectively (data not shown). The results reflect higher levels ofexpression and/or secretion of recombinant 2G11 and 18H6 dengueantigens, expressed from pMaxVax10.1_(12G11) and pMaxVax10.1_(18H6),respectively. These results evidence the improved secretion and/orexpression of recombinant dengue virus antigens of the invention.

Example 14

[0692] This example demonstrates the production of recombinant C15/fulllength prM/full length E CO dengue nucleotide sequences and recombinantfusion proteins encoded therefrom using recursive sequence recombinationmethods. Libraries comprising such recombinant nucleotide sequences werealso generated.

[0693] Den-1PRM15/tE CO, Den-2 PRM15/tE CO, Den-3 PRM15/tE CO, and Den-4PRM15/tE CO nucleotide sequences were extended using the techniques ofExample 12 to generate Den-1 C15/full length prM/full E CO, Den-2C15/prM/full length E CO, Den-3 C15/full length prM/full length E CO,and Den-4 C15/full length prM/full length E CO dengue nucleotidesequences, respectively. The plasmid DNA for each of these 4 extendedantigen-encoding sequences was isolated from E. coli amplification,purified, and used as starting sequence material in recursive sequencerecombination as described in Example 1. Recombinant nucleic acids wereisolated by appropriate rescue primers and ligated into pMaxVax10.1vectors. Library transfections of E. coli were performed as described inExample 2.

Example 15

[0694] This example demonstrates the ability of a recombinant denguevirus antigen of the invention comprising a recombinant fusion proteincomprising a recombinant C15 signal peptide/full length prM protein/fulllength E protein, recombinant full length prM protein/full length Eprotein, or recombinant full length M/full length E protein to induce,enhance, or modulate production of antibodies that react with (or bindor specifically bind to) dengue viruses of multiple serotypes in vivoand/or ex vivo.

[0695] Libraries of recombinant nucleic acid sequences were generated byrecursive sequence recombination using as parental sequences four humancodon-optimized dengue virus nucleotide sequences encoding WT Den-1,Den-2, Den-3, and Den-4 polypeptides, respectively. Each such parentalnucleotide sequence comprised the following: a nucleic acid encoding amethionine, a nucleotide sequence encoding the last 15 amino acidresidues of the C terminal of the capsid (C) protein of the respectiveWT dengue virus (which served as a signal sequence), a nucleotidesequence encoding a full length prM sequence of the respective WT denguevirus, and a nucleotide sequence encoding the full length E protein ofsaid respective WT dengue virus. Each such parent encoded a recombinantfusion protein comprising a Met residue at the N terminal, a recombinantamino acid sequence of 15 amino acid residues that served as a signalsequence, a recombinant full length prM protein, and a recombinant Eprotein.

[0696] Dot blot analyses of cell culture medium supernatants, whereineach supernatant obtained from 293 cells transfected with a particularpMaxVax10.1 vector comprising a specific shuffled nucleotide sequenceobtained via shuffling of the C15/full prM/full envelope dengue virusparental human codon optimized nucleotide sequences, were performed.Analyses indicated the following recombinant C15/full prM/full E dengueantigen clones were expressed, secreted, and recognized by a mix ofDEN-1, DEN-2, DEN-3, and DEN-4 mouse antibodies: 21C1 (SEQ ID NO:142),23C12 (SEQ ID NO:143), 23D5 (SEQ ID NO:144), 23F5 (SEQ ID NO:145), 23G3(SEQ ID NO:146), 23H7 (SEQ ID NO:148), 25B6 (SEQ ID NO:236), 25B10 (SEQID NO:237), 25D4 (SEQ ID NO:238), 25E11 (SEQ ID NO:239), 25H4 (SEQ IDNO:240), 27A11 (SEQ ID NO:241), 27G6 (SEQ ID NO:242), 28A11 (SEQ IDNO:243), 28C1 (SEQ ID NO:244), 28D11 (SEQ ID NO:245), 28E12 (SEQ IDNO:246), 28F9 (SEQ ID NO:247), 28H3 (SEQ ID NO:248), 28H9(SEQ IDNO:249). The pMaxVax10.1 DNA plasmid vectors comprising the shuffledC15/full length prM/full length E protein nucleotide sequences forclones 21C1, 23C12, 23D5, 23F5, 23G3, and 23H7 were isolated, purified,and injected into mice, along with pMaxVax10.1_(null), following the DNAinjection regimen described in Example 5. The experiments were performedin triplicate (i.e., three mice received repeated injections of each ofthe indicated plasmids). Sera from the mice were obtained at appropriatetimes (e.g., 28, 55, and 76 days after initial DNA injection (day 0)),and analyzed by ELISA, as described above, using DEN-1, DEN-2, DEN-3,and DEN-4 virus coated ELISA plates. Average OD values observed in theELISA assays were calculated for each DNA vector tested. The results ofthese calculations are set forth for day 76 (“d76”) antisera in FIG. 10.

[0697]FIG. 10 shows that all of these recombinant full length prM/fulllength E dengue antigens (Ag) (expressed with C15 signal peptide)induced production of populations of antibodies in vivo that reactedwith all 4 DEN serotypes in vitro. OD values obtained in ELISA assayswith pMaxVax10.1_(23G3) and pMaxVax10.1_(23H7) were the highest andsimilarly strong in all 4 assays with the 4 dengue virus serotypes. Thesignal peptide of the C15/full prM/full E dengue antigens is typicallycleaved and thus similar or equivalent immune response andimmune-stimulating results are believed produced with full prM/full Edengue antigen polypeptides.

[0698] The results of this experiment demonstrate that recombinantantigens of the invention comprising recombinant C15/full lengthprM/full length E protein sequences are capable of inducing, enhancing,and/or modulating the production of antibodies that react with (or bindor specifically bind to) one, two three, or 4 dengue virus serotypes inin vivo, ex vivo, and/or in vitro methods of the invention.

Example 16

[0699] This example demonstrates the secretion and/or expressioncharacteristics of recombinant dengue antigens of the invention whichcomprise recombinant fusion proteins comprising, e.g., a recombinant C15signal peptide/full length prM/full length E protein sequence, asdetermined by Western blot analysis. The invention also includesrecombinant full length prM/full length E protein sequences andrecombinant full length M/full length E protein sequences, which areproduced by enzymatic cleavage of the C15 signal peptide from a C15signal peptide/full length prM/full length E protein sequence andenzymatic cleavage of a “pr” segment from a full length prM/full lengthE protein sequence, respectively.

[0700] Individual 293 cell cultures were each transfected with apMaxVax10.1 vector comprising a nucleotide sequence corresponding to oneof the following eight representative recombinant clones: 23H7, 23G3,23F5, 23D5, 23C12, 23A11, 21C1, and 21B4. The nucleic acid sequence ofeach such clone was generated and identified by the recursive sequencerecombination (see, e.g., Example 15) and selected screening assays.Clones 23A1 and 21B4 were negative in an ELISA analysis performed by thetechniques described in Examples 1 and 15. ELISA analysis indicated thatthe remaining six clones secreted and/or expressed recombinant antigensthat induced production of antibodies that reacted with all four denguevirus serotypes in vivo (FIG. 10). After an appropriate incubationperiod under transgene expression-permissive conditions, theunconcentrated supernatant (cell-free portion of medium) from thetransfected 293 cell cultures was collected, separated by polyacrylamideelectrophoresis, and blotted onto an appropriate membrane for Westernblot analysis as described in Example 1, using anti-DEN-1, DEN-3, andDEN-4 antibodies from mouse ascitic fluid. The nitrocellulose membranebound proteins were incubated with antibodies from mouse ascitic fluidfor DEN-1, DEN-3, and DEN-4. The recombinant antigens (produced by DNAshuffling and appropriate screening/selection procedures using C15/fulllength prM/full length E protein-encoding nucleotide sequence formats)that were positive for production of a multivalent antibody response invivo (e.g., 23H7, 23G3, 23F5, 23D5, 23C12, 21C1) were associated with atleast one visible band in the Western blot (data not shown), whereas theclones that did not induce such an antibody response (e.g., 23A11 and21B4) were not associated with any significant bands in the Westernblot, suggesting poor expression and/or secretion of proteins encoded by23A11 and 21B4 clones as compared with the recombinant antigens 23H7,23G3, 23F5, 23D5, 23C12, 21C1.

[0701] Additionally, bands corresponding to multimers (e.g., full lengthE protein dimers, trimers, and/or other multimers and/or misfoldedprotein complexes) were observed in the Western blot (data not shown).Since wild-type E protein is believed to form homodimeric rods on thesurface of the virion (FIELDS VIROLOGY, supra), the appearance of bandscorresponding to the size of E protein dimers by Western Blot indicatesthat the majority of the recombinant envelope proteins are correctlyfolding. Multimers of other polypeptides of the invention include, butare not limited to, e.g., C15/full length prM/full length E proteinmultimers; full length prM/full length E protein multimers; full lengthM/full length E protein multimers; PRM15/full length E proteinmultimers; PRM15/truncated E protein multimers).

[0702] The results of these experiments demonstrate that select nucleicacid sequences of the invention can be used to effectively expressrecombinant dengue antigens as secreted proteins.

Example 17

[0703] This example demonstrates the sequence diversity observed invarious nucleic acids of the invention as compared to correspondingwild-type sequences.

[0704] The amino acid sequences of 16B4, 16G11, 18H2, 18H6, 18E11,18E10, 18E9, 14G10, and 18D7, each of which comprised a PRM15/truncatedE format, were determined and compared to the amino acid sequences ofeach of the WT parental polypeptides, DEN-1 PRM15/truncated E, DEN-2PRM15/truncated E, DEN-3 PRM15/truncated E, and DEN-4 PRM15/truncated E.The results of these analyses are provided in FIG. 11. As shown in thisfigure, these recombinant PRM15/truncated E protein antigens of theinvention exhibited complex sequence diversity, comprising multipleamino acid fragments or segments, each such fragment or segmentcomprising one or more amino acid residues of the four parental WTPRM15/tE protein dengue virus antigen sequences.

[0705] Recombinant polypeptide antigens corresponding to clones 23G3,23H7, 23F5, 23C12, 23D5, 21C1, 21B4, 23A11, 5/21-D1, and 6E12-D4, eachof which comprised a C15/full length prM/full length E protein format,were similarly sequenced and compared with respect to amino acidsequences of each of the DEN-1, DEN-2, DEN-3, and DEN-4 C15/full lengthprM/full length E fusion proteins. These antigens were similarly foundto have complex sequence diversity, comprising multiple amino acidfragments or segments, each such fragment or segment comprising one ormore amino acid residues of the four parental WT C15/full prM/full Eprotein dengue virus antigen sequences (data not shown).

Example 18

[0706] This example demonstrates, among other things, the ability ofrecombinant dengue virus antigens of the invention to induce or promotea protective immune response(s) in vivo in a subject, and administeredex vivo in tissue or cells of a subject, and/or in vitro in a populationof cells. In this example, such immune response is induced or promotedupon expression of a recombinant dengue virus antigen of the inventionfrom a plasmid vector comprising a nucleotide sequence encoding suchantigen; the amount of plasmid vector administered is that sufficient toproduce an immunogenic amount of the recombinant antigen. A protectiveimmune response(s) can be similarly induced or promoted by in vivo, exvivo, and/or in vitro administration to a subject of a an immunogenicamount of a polypeptide comprising such recombinant dengue virusantigen.

[0707] Dengue viruses do not typically induce clinical syndromes inadult mice. However, dengue viruses injected into the cerebellum ofyoung mice (e.g., about 3-6 weeks old) cause paralyses, encephalitis anddeath. Such a mouse model can thus be used to evaluate the in vivoprotective efficacy of sera produced in mice immunized with recombinantpolypeptides of the invention or nucleic acids encoding such recombinantpolypeptides.

[0708] Mice were immunized with 100 μg plasmid DNA of one of thefollowing vectors: pMaxVax10.1_(18H6), pMaxVax10.1_(2G11-D4),pMaxVax10.1_(6E12-D4), a mix ofpMaxVax10.1_(18H6)/pMaxVax10.1_(2G11-D4)/pMaxVax10.1_(6E12-D4),pMaxVax10.1_(Den-2PRM15/tE CO), pMaxVax10.1_(Den-2 C15/fullprM/full E),pMaxVax10.1_(Den-3C15/fullprM/full E P), a mix of four WTDEN-1-4_(pRM15/tE CO), a mix of four WT DEN-1-4_(C15/fullprM/full E),and, pMaxVax10.1_(null) (termed “vector control” in FIG. 12); one groupof mice was immunized with PBS buffer only. The nucleotide sequencescorresponding to clones 18H6, 2G11-D4, and 6E12-D4 were SEQ ID NOS:235,204, and 202, respectively; such sequences were generated using PRM15/tE(for 18H6) and C15/fullprM/full E (for 2G11-D4 and 6E12-D4) parentalnucleotide sequences. Injections were repeated at day 10 after initialDNA injection. At 21 days after initial DNA injection, the mice werechallenged with intracerebral injections of 100 LD₅₀ of DEN-2 virusparticles. The injection and infection experiments were repeated in 5different mice for each vector or control tested.

[0709] The mice were observed for 28 days after challenge for signs ofparalyses, encephalitis and/or death. A graph of the survival rates inthe first 28 days after challenge is provided in FIG. 12. The resultsreported in FIG. 12 demonstrate that the injection of a plasmid encodingeither the 18H6 and WT DEN-2 polypeptide antigen (each in PRM15/tEformat), or 6E12-D4 and WT DEN-2 (each in C15/full prM/full E format),as well a composition or mixture of the three recombinant antigens(18H6, 2G11-D4, and 6E12-4) in PBS was able to induce, enhance, orpromote a protective immune response(s) against challenge with DEN-2virus in vivo. At 28 days after DEN-2 virus challenge, all of the miceimmunized with the above-listed recombinant clones were normal and thusprotected. Mice immunized with either of the WT DEN-2 polypeptides (ineither PRM/tE or C15/full prM/full E format) were also protected asobserved 28 days after DEN-2 virus challenge. In contrast, of the miceinjected with a tetravalent mix of the four WT DEN-1-4 antigens in thePRM/tE or C15/full prM/full E format only 50% were protected as observed28 days after DEN-2 virus challenge. Of the mice injected with DEN-3C15/full prM/full E antigen, only 50% were protected as observed 28 daysafter DEN-2 virus challenge. All of the PBS-immunized mice, and 75% ofthe mice that received injections of the vector control (pMV10.1 weredead 12 days after challenge with DEN-2 virus. The results of theseexperiments demonstrate that such recombinant polypeptide antigens ofthe invention (and nucleic acids encoding such recombinant antigens) arecapable of inducing, promoting, and/or enhancing a protective immuneresponse(s) against at least wild-type Den-2 virus in vivo. Analogousexperiments can be performed using other animal models, if desired.

[0710] Analogous experiments are performed using mice (or other animalmodels) immunized with these recombinant antigens and challenged withDen-1, Den-3, and/or Den-4 viruses to assess the immune response(s)induced, promoted, or modulated by such recombinant antigens against oneor more of these WT dengue viruses.

[0711] Similar experiments are performed using mice or other animalmodels immunized with other recombinant antigens of the invention,and/or nucleic acids encoding such antigens, and challenged with atleast one of the four WT dengue virus serotypes.

Example 19

[0712] This example illustrates the preparation and use of recombinantantigens of the invention, including, e.g., tetravalent antigens, asdiagnostic antigens or screening antigens (e.g., diagnostic tools) inmethods to detect, diagnose, screen for, and/or identify the presence ofantibodies against at least one, preferably two or more, and even morepreferably, all 4 WT dengue virus serotypes in a sample, such as abiological sample from a mammal. Such recombinant antigens can be usedfor serum analyses either coated to a microtiter plate or spotted on asuitable membrane (nitrocellulose, nylon) for dot blot analysis or to adip-stick membrane. Such microtiter plates or membranes, or dip-stickscan be coated with the recombinant antigens directly from thesupernatant of transfected cells.

[0713] Four of the tetravalent antigens of the invention, correspondingto clones 2/7, 5/21, 2G11, and 6E12 as described in Example 5, eachhaving a recombinant PRM15/tE protein format, were selected to test asdiagnostic antigens. Recombinant C15/full prM/full E polypeptideantigens, which are believed to form VLPs, can also be used (e.g.,2/7-D1, 5/21-D1, 2G11-D4, and 6E12-D4). The feasibility of use of arecombinant antigen as a diagnostic antigen or diagnostic or screeningtool in diagnostic or screening methods of the invention for thedetection, diagnosis, screening or identification of the presence ofantibodies against one or more of the 4 WT dengue virus serotypes in asample was demonstrated by a dot blot assay. Specifically, human 293cells were transfected with pMaxVax10.1 plasmids coding for each of thefour selected tetravalent antigens. Three days after transfection, themedia supernatants were harvested and spotted in serial dilution of10-110 μl on nitrocellulose membranes. To evaluate the sensitivity ofthe assay, sera from mice (or sera from other mammals, such as humans)infected with dengue viruses were used in a 1:2000 dilution in PBS todetect or screen for type-specific antibodies. For detection orscreening, the membranes were first incubated with the test sera andsubsequently with secondary antibodies linked to a detection reagent(HRP (horseradish peroxidase), AP (alkaline phosphatase), or FITC(Fluorescein isothiocyanate)). The dot blot of the spotted mediasupernatants demonstrates that each of these recombinant polypeptidevariants was well secreted (FIG. 13A) and recognized by all 4type-specific anti-DEN antisera (FIG. 13B); thus, these clones can beused to detect or screen for serum antibodies against any of the 4 WTDEN serotypes. Moreover, as illustrated in FIGS. 13A and 13B, as littleas about 10 μl of the crude supernatants from cell culture medium perdot in the assay was sufficient for detection of type-specific DENantibodies in the sera, which makes manufacturing diagnostic dot blotsusing the recombinant dengue antigens (including, e.g., recombinanttetravalent dengue antigens) easy and inexpensive.

[0714] The results of these experiments demonstrate that even a verysmall amount of a recombinant dengue virus antigen of the invention (orcomposition thereof) provides a convenient substrate for analysis ofbiological samples obtained from subjects, e.g., mammals, includinghumans, for the presence or absence of anti-dengue virus antibodiesagainst one or more virus serotypes. The present invention includesmethods for detecting and/or screening for the presence of one or moretype-specific antibodies in sera of dengue virus infected mammals,including humans. Such methods include those described above. Inaddition, the recombinant antigens of the invention (and nucleic acidsencoding them) can be used in other diagnostic assays, such as, e.g.,plate ELISA and dip stick assays, for detection of one or moretype-specific antibodies in sera of dengue virus infected mammals.

[0715] The present invention also includes diagnostic dot blotscomprising one or more of the recombinant dengue antigens of theinventions, and preferably including one or more of those recombinantdengue antigens of the invention that react (or bind or specificallybind to) antibodies of two or more, and preferably all four dengue virusserotypes. The invention further provides kits comprising one or moresuch diagnostic dot blots and/or one or more such diagnostic recombinantantigens.

Example 20

[0716] This example illustrates the dot blot analysis of a nucleotidelibrary comprising recombinant C15/full length prM/full length E proteinnucleotide sequences and selection from such libraries of thoserecombinant nucleotides expressing recombinant antigens (in recombinantC15/full length prM/full length E protein format or full length prM/fulllength E protein format) that exhibit improved secretion and/orexpression in mammalian cells as compared to the secretion and/orexpression WT prM/E antigens, respectively, in such mammalian cells.

[0717] Library transfections of E. coli and subsequent transfection of293 cells in a 96 well cell culture format using plasmids comprisingisolated clone DNA sequences encoding antigens in C15/full lengthprM/full length E protein format (or full length prM/full length Eprotein format) were carried out in accordance with the techniquesdescribed in Examples 2 and 14. Plasmids comprising a nucleotidesequence of the same format—C15/full length prM/full length E fusionprotein-encoding nucleotide sequence (or a full length prM/full length Eprotein-encoding nucleotide sequence)—of one of each of the 4 WT denguevirus serotypes were used as controls in 293 cell transfections. Thecell-free aspirated supernatant obtained from such 293 cell culturesafter an appropriate period for recombinant antigen expression are thenbound to membranes by application of standard dot blot techniques inaccordance with applicable manufacturer instructions. The dot blotmembranes are incubated with anti-DEN-1, DEN-2, DEN-3, and DEN-4antibodies from mouse ascitic fluid and appropriate enzyme-conjugatedsecondary antibodies in accordance with the methods described in Example1 for Western blots. Clones that indicate higher levels of secretionand/or expression than the control WT dengue C15/full prM/fullE-encoding nucleic acids (WT dengue full prM/full E-encoding nucleicacids), as shown by the presence of a more intense signal on the dotblot, are then selected for further cloning and analysis.

[0718] This experiment demonstrates an exemplary strategy for analyzinga library of recombinant nucleic acids encoding recombinant dengueantigens, which have an amino acid sequence similar in size to a fulllength prM/full E fusion protein, for those antigens exhibiting improvedor enhanced levels of secretion and/or expression in mammalian cellsthan corresponding WT dengue antigens of similar size.

Example 21

[0719] This example illustrates the production of recombinant dengueantigens each having an amino acid sequence identical in length to orsubstantially similar or substantially identical in length (e.g., atleast about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably at leastabout 90%, 91%, 92%, 93%, or 94%, and more preferably at least about 95%(e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% identity in length) to afusion protein comprising a C15 signal sequence, full length prMprotein, and full length E protein.

[0720] The nucleotide sequences of clone 25B10 (SEQ ID NO:259) (inC15/full prM/full E protein format) and clone 16G11 (PRM15/tE format)(SEQ ID NO:187) were digested with appropriate restriction enzymes, andthe nucleotide sequence of clone 16G11 was extended at its C terminus (Cterminus of its truncated E protein) with a sufficient and appropriatecodon optimized nucleotide segment of the C terminus of the E protein of25B10 (see SEQ ID NO:259) and was extended at its N terminus (N terminusof its PRM15 signal peptide) with a sufficient and appropriate codonoptimized nucleotide segment of the N terminus of the C15/full prM of25B10 (see SEQ ID NO:259) to make a recombinant clone having a C15/fulllength prM/full length E format, as described in Example 12.

[0721] Similarly, the nucleotide sequences of recombinant clone 25B10(SEQ ID NO: 259) (in C15/full prM/full E protein format) and recombinantclone 18H6 (PRM15/tE format) (SEQ ID NO:235) were digested withappropriate restriction enzymes, and the nucleotide sequence of clone18H6 was extended at the C terminus of its truncated E protein with asufficient and appropriate codon optimized nucleotide segment of the Cterminus of the E protein of 25B10 (see SEQ ID NO:259) and was extendedat the N terminus of its PRM15 with a sufficient and appropriate codonoptimized nucleotide segment of the N terminus of the C15/full prM of25B10 (observed in SEQ ID NO:259) to make a recombinant clone having aC15/full length prM/full length E format, as described in Example 12.Similarly, the nucleotide sequences of wild-type DEN-4 C15/full lengthprM/full length E protein (SEQ ID NO:234) and recombinant clones 16G11(PRM15/tE format) (SEQ ID NO:187) and 18H6 (PRM15/tE format) (SEQ IDNO:235) were digested with appropriate restriction enzymes, and thenucleotide sequences of each of 16G11 and 18H6 were extended at the Cterminus of the truncated E protein with sufficient and appropriatecodon optimized nucleotide segment of the C terminus sequence of the Eprotein-encoding sequence of DEN-4 C15/full prM/full E protein-encodingnucleotide sequence (SEQ ID NO:234), and at the N terminus of the PRM15with a sufficient and appropriate codon optimized nucleotide segment ofthe N terminus sequence of the C15/prM of DEN-4 C15/full prM/full Eprotein-encoding nucleotide sequence to make respective recombinantclones having C15/full length prM/full length E formats, as in Example12. The resulting recombinant nucleic acids were named 16G11-25B10ext(SEQ ID NO:255) (i.e., 16G11 in PRM15/tE format extended to C15/fullprM/full E format with appropriate nucleotide sequences of clone 25B10);16G11-D4 (SEQ ID NO:254) (i.e., 16G11 in PRM15/tE format extended toC15/full prM/full E format with appropriate nucleotide sequences of WTDen-4); 18H6-25B10ext (SEQ ID NO:257) (i.e., 18H6 in PRM15/tE formatextended to C15/full prM/full E format with appropriate sequences ofclone 25B10); and 18H6-D4 (SEQ ID NO:256) (i.e., 18H6 in PRM15/tE formatextended to C15/full prM/full E format with appropriate sequences of WTDen-4). These extended nucleic acid sequences were amplified and clonedinto pMaxVax10.1 vectors, as described above. The correspondingrecombinant polypeptide sequences for 16G11-25B10ext, 16G11-D4,18H6-25B10ext, and 18H6-D4 are SEQ ID NOS:255, 254, 257, and 256,respectively. These amplified plasmids were analyzed in vivo byinjection of mice and subsequent ELISA assays essentially as describedin Example 15. The recombinant antigens of clones 25B10, 16G11-25B10ext,16G11-D4, 18H6-25B10ext, and 18H6-D4 all induced antibodies in mice thatstrongly cross-reacted with all 4 dengue virus sero-types in ELISAassays (data not shown).

Example 22

[0722] This example illustrates the use of ELISpot assay (Enzyme-LinkedImmunoSPOT) to measure the number of T lymphocytes (T cells) secretinginterferon gamma, as a marker of the T cell activating effects ofpolypeptides of the invention.

[0723] Subjects selected from an appropriate mammalian model for testingdengue virus vaccines (including, e.g., but not limited to, inbred mice,monkeys, or chimpanzees) or human patients (e.g., in a phase I clinicaltrial) are injected with a particular pMaxVax10.1 DNA plasmid vector(about 0.5-2 mg DNA plasmid vector in about 0.5-1 ml of anendotoxin-free carrier (e.g., PBS or another suitable saline buffer,0.1M NaCl, or any suitable carrier which maintains the pH of thesolution at about 7.4) injection volume for, e.g., humans or monkeys;plasmid amounts/compositions similar to those used in theabove-described experiments for in vivo mice experiments are suitablefor mice), wherein each vector comprises a particular recombinantantigen-encoding nucleotide sequence of the invention. For example, eachsubject is injected with a pMaxVax10.1 DNA plasmid vector that comprisesone of the following: 1) a recombinant nucleotide sequence that encodesa PRM15/tE fusion protein (e.g., nucleotide sequences having SEQ IDNOS:157 (2G11), 158 (5/21), 159 (6E12), 185 (16B4), 187 (16G11), 172(18E11), 200 (18H2), 235 (18H6)); 2) a recombinant nucleotide sequencethat encodes a C15 signal peptide/full length prM protein/tE fusionprotein (e.g., nucleotide sequences having SEQ ID NOS:254 (clone16G11-D4), 255 (clone 16G11-25B10ext), 256 (clone 18H6-D4), 257 (clone18H6-25B10ext)) (see, e.g., methods described in Examples 1 and 12); oranother recombinant nucleotide sequence of the invention. Optionally,but preferably, initial DNA plasmid administration is followed byboosting with an immunogenic amount of the recombinant polypeptideencoded by the same nucleic acid included in the plasmid vector(delivered initially) or by re-administration of the same pMaxVax10.1vector at about 14-28 days after initial DNA plasmid injection.Preferably, a second (polypeptide) protein boost (using the samerecombinant polypeptide) and/or DNA plasmid vector boost (using the sameplasmid vector that encodes the same recombinant polypeptide) isperformed between 2-6 months after the initial infection. One group ofcontrol subjects receives no injection. Another group of controlsubjects receives injections of pMaxVax 10.1_(null). Experiments arepreferably performed with multiple subjects for each plasmid constructtested.

[0724] Sera are collected from the subjects at appropriate test times(e.g., about 60 days, 120 days, or 180 days after initial DNA plasmidinjection, preferably including a time falling at least 2-4 weeks afterthe last recombinant polypeptide antigen/DNA vector boost) and T cellsisolated by methods known in the art. For example, in human patients ina clinical study, peripheral blood mononuclear cells (PBMC) can beisolated from human blood by centrifugation over Histopaque-1077 (Sigma,St. Louis, Mo.) (using Ficoll gradient separation). T cells are isolatedand purified either by staining the cells with anti-human CD2 monoclonalantibodies (mAbs) and sorting for CD2 positive (CD2⁺) cells using a FACSVantage SE or removing cells that stained with mAbs specific for CD 14,CD20, CD56 and CD94 by magnetic beads (e.g., Dynabeads, Dynal, LakeSuccess, N.Y.) following the manufacturer's instructions. Antibodiesuseful in such techniques can be purchased from Pharmigen (San Diego,Calif.). Magnetic separation of the T cells using Dynal Dynabeads isperformed by first labeling PBMCs with pure monoclonal antibodiesagainst CD14, CD20, CD56 and CD94, then labeling the cells with Sheepanti-mouse Dynabeads according to manufacturer's instructions. Non-Tcells are removed by depleting with a magnet. The purity of the T cellsshould be 96-99% when analyzed by staining with anti-CD3 mAbs purchasedfrom Pharmigen (San Diego, Calif.).

[0725] A 96 well ELISpot plate is coated with antibody (immunoglobulin(Ig)) that is specific for IFN-gamma. For example, NIB42 captureantibody (Pharmigen) can be used for plate coating. Other captureantibodies that can be used are known in the art. The antibody (Ig)binds to the nitrocellulose base of the ELISpot plate.

[0726] The activated T cells are transferred to the ELISpot plate, andIFN-gamma is released during the incubation period. IFN-gamma releasedlocally around T cells is bound, and therefore is “captured” by theIFN-gamma specific antibody. The cells and any excess cytokines arewashed off. A second antibody that is also specific for IFN-gamma isadded. This antibody is coupled to an enzyme that is capable ofconverting a substrate into an insoluble colored product. For example,4S.B3 biotinylated detection antibody (Pharmigen) can be used toquantify IFN-gamma production. Other detection antibodies that can beused are known in the art. The plates are washed once more, and theenzyme substrate is added. The substrate is converted into the insolubleproduct, forming spots of color that represent the areas of capturedcytokines that were secreted by adjacent T cells. The colored spots arecounted using a microscope or digital-imaging system such as an ELISpotreader (such as the ELISpot reader sold by Scanalytics, Inc.) byfollowing manufacturer's instructions.

[0727] T cells obtained from sera of subjects that have been injectedwith a pMaxVax10.1 vector comprising a recombinant nucleic acid sequenceof the invention exhibit a significantly higher level of IFN-gammaexpression than T cells obtained from the sera of subjected injectedwith pMaxVax10.1_(null) or receiving no DNA injection.

[0728] This example illustrates the use of ELISpot to quantify thenumber of circulating antigen-specific CD8+ T cells. This example alsodemonstrates the antigen-specific increase in T cell activity associatedwith administering a DNA sequence encoding a recombinant polypeptide ofthe invention to a subject host, e.g., mammal.

Example 23

[0729] This example demonstrates ELISPot analysis of T cell associatedIFN-gamma expression induced or modulated by recombinant polypeptides ofthe invention.

[0730] PBMCs are isolated from a mammal model or human and stimulatedwith one or more recombinant antigens of the invention, such as, e.g.,but not limited to, SEQ ID NOS:66 (clone 2G11), 67 (5/21), 69 (6E12), 89(16B4), 93 (clone 16G11), 108 (18E11), 109 (18H2), 110 (18H6), all ofwhich are of the PRM15/tE format, or SEQ ID NOS:254 (clone 16G11-D4),256 (16G11-25B10ext), 257 (18H6-D4), and/or 258 (18H6-25B10ext), all ofwhich are of the C15/full prM/full E format, for about 6-24 hours andthen mixed with enzymatically-tagged cytokine antibodies. The mixture isthen chemically treated so that bound antibody-cell complexes (i.e.,cytokine-secreting cells) are stained blue.

[0731] A suitable IFN-gamma ELISpot assay that can be used forquantitative IFN-gamma ELISPot analysis is commercially availablethrough Biosource International, Camarrillo, Calif. The assay isperformed according to the manufacturer's instructions and usingstandard techniques. For example, enumeration of IFN-gamma secretingcells in single cell suspension is performed by adding 50 μl of dilutedcoating antibody to each well followed by the addition of 50 μl of PBS.Each well is incubated overnight at 4° C. Samples are then aspirated andwashed 5 to 10 times with wash buffer. 200 μl of post-coating solutionis added into each well and wells are incubated 1 hour at 37° C. orovernight at 4° C. The wells are aspirated and not washed. Wells are 100μl of pre-stimulated single cell preparation are added into the wells.The plate is covered with the plate cover and incubated for 5 hours at37° C. in a humidified atmosphere containing 7% CO₂. The wells areaspirated, 200 μl ice-cold deionized water is added, and the plate isplaced for 10 min on melting ice. The wells are washed 10 times withPBS. 100 μl of diluted biotinylated Antibody solution is added, theplate is covered and incubated for 1 hour at 37° C. or overnight at 4°C. The wells are aspirated and washed 5 to 10 times with PBS. 50 μl ofdiluted—labeled anti-biotin antibody solution (GABA) is added to eachwell. The plate is covered and incubated 1 hour at 37° C. The wells areaspirated and washed 5 to 10 times. 30 μl of activator solution is addedto each well. The spot development is followed by light microscopy. Whenclear spots have developed, the reactions are stopped by rinsing thewells with distilled water. The results using recombinant clones in thePRM15/tE fusion protein format and C15/full length prM/full length Efusion protein format are compared with results obtained with subjectsimmunized with wild type dengue PRM15/tE fusion proteins and C15/fulllength prM/full length E fusion proteins. Results are also compared withsubjects that were not immunized with any dengue antigen.

[0732] The results of such ELISpot analysis indicate that recombinantpolypeptides of the invention are able to induce and/or modulateIFN-gamma expression from T cells. The present invention also providesalternative techniques for conducting ELISpot assays with recombinantpolypeptides and/or polynucleotides of the invention.

Example 24

[0733] This example demonstrates a T-cell proliferation assay to measurethe proliferation of a patient's T cells in response to stimulation withrecombinant polypeptide antigens of the present invention.

[0734] T cells in a suitable subject (preferably a human patient) arestimulated with pMaxVax10.1 vectors as described in Example 22, followedby removal of a whole blood-based sample and separation of specificT-cell subsets. Monoclonal antibodies attached to paramagnetic particlesrecognize specific cell surface markers, and T cells are isolated whenthe sample is placed in a magnetic field. Several signs of T cellactivation can be measured by such a technique. For example, increasedintracellular ATP accumulation is associated with T-cell activation, soa luminescent ATP assay employing firefly luciferase can be used tomeasure the level of activation among isolated cells (an example of acommercially suitable assay in this respect is the Luminetics Assay,available through Cylex).

[0735] Additionally or alternatively, T cell proliferation is measuredby ³H-thymidine incorporation. Briefly, 293 cells (or COS-7 cells orother cells of interest) are transfected with a pMaxVax10.1 plasmidvector encoding a recombinant dengue antigen of the invention, e.g., arecombinant tetravalent dengue polypeptide antigen (as described, e.g.,in Example 22) or a control plasmid vector lacking an insert (e.g.,pMaxVax10.1_(null) vector). The Effectine HTP (Qiagen, Valencia, Calif.)96-well transfection method is used for plasmid transfections accordingto manufacturer's instructions.

[0736] Twenty-four hours after transfection, approximately 5×10⁴purified T cells (purified as described, e.g., in Example 21) arecultured in triplicate in the presence of irradiated (5000 radians(rads)) transfectants and soluble anti-human CD3 mAbs (5 μg/mL) in a96-well plate culture format (e.g., the system commercially availablethrough VWR, Westchester, Pa.) at 37° C. in a humidified atmospherecontaining 5% CO₂ in Yssel's medium supplemented with 10% FCS (200μl/well) for a total of 3 days (72 hours). 1 microCurie (μCi)/well of³H-thymidine (Amersham, Piscataway, N.J.) is added by pulsing to thecell cultures during the last 8 hours of the culture period, and thecells are harvested for counting onto filter paper by a cell harvester(Tomtec, Hamden, Conn.). ³H-thymidine uptake/incorporation in thecultured cells is determined by measuring the radioactivity on the driedfilters using a MicroBeta scintillation counter (Wallac, Turku, Finland)according to the manufacturer's instructions. Proliferation of T cellsis expressed as the mean counts per minute (cpm) of triplicate wells.

[0737] At least some of the recombinant polypeptide antigen-encodingplasmids of the invention (e.g., recombinant tetravalent polypeptideantigen-encoding plasmids of the invention) induce T cell responsesand/or T cell proliferation at levels significantly higher than thoseseen in a plasmid control and/or at least as high a level as observedwith plasmids encoding a WT dengue virus fusion protein having about thesame length or substantially similar or substantially identical length(e.g., at least about 75%, 80%, 85%, 86%, 87%, 88% or 89%, preferably atleast about 90%, 91%, 92%, 93%, or 94%, and more preferably at leastabout 95% (e.g., about 87-95%), 96% 97%, 98%, 99%, 99.5% identity inlength) as (e.g., if the plasmid encodes a recombinant polypeptideantigen comprising a PRM15/tE fusion protein, a plasmid encoding a WTdengue polypeptide comprising a DEN 1, 2, 3, or 4 PRM15/tE fusionprotein is used for comparison; if the plasmid encodes a recombinantpolypeptide antigen comprising a C15/full length prM/full length Efusion protein, a plasmid encoding a WT dengue polypeptide comprising aDEN 1, 2, 3, or 4 C15/full length prM/full length E fusion protein isused for comparison). Plasmid vectors encoding a recombinantPRM15/tE-encoding sequence or recombinant C15/full length prM/fulllength E-encoding sequence that induce or promote T cell proliferationand/or activation at a level significantly above that induced orpromoted by the plasmid vector control (or other control), andpreferably at a level at least about similar or equivalent to thatinduced or promoted by a pMaxVax10.1 vector comprising a wild-typedengue virus PRM15/tE-encoding sequence or C15/full length prM/fulllength E-encoding sequence, respectively, can be identified and selectedor isolated for further characterization (e.g., sequencing, effects inother T cell activity assays, and/or diversity analysis).

[0738] This experiment demonstrates assays suitable for assessing T-cellproliferation effects of recombinant polypeptide antigens of theinvention. This experiment also demonstrates that at least somerecombinant antigens of the invention induce or promote T cellproliferation and/or T cell activation effects in mammalian cells.

Example 25

[0739] This example illustrates the use of a cytotoxicity assay toquantify the amount of T cells expressing a recombinant dengue virusantigen of the invention in a particular sample.

[0740] Populations of 293 cells are transfected with a pMaxVax10.1plasmid vector encoding a recombinant polypeptide antigen of theinvention, with a pMaxVax10.1_(null) plasmid vector (control), or withnothing as described in Examples 22. The transfected cells are labeledwith a radioactive isotope of chromium (⁵¹Cr). A subject's T cells(e.g., human T cells) that have been isolated and purified as describedin Example 21 are obtained, mixed with the target cells, and the mixtureis incubated for several hours. Lysis of antigen-expressing cellsreleases ⁵¹Cr into the medium. Recombinant antigen-specific lysis iscalculated by comparing lysis of 293 cells expressing a recombinantantigen of the invention or control antigen in the presence or absenceof the subject's effector cells. The results of such experiments areexpressed as a percentage of cells exhibiting recombinantantigen-specific lysis. The percentage of cells exhibiting recombinantantigen-specific lysis in such experiments demonstrates the recombinantantigens of the invention are capable of inducing a significantcytotoxic immune response in a subject. This example provides a methodof measuring the amount (number) of T cells expressing recombinantdengue virus specific antigens.

Example 26

[0741] This example illustrates the use of a tetramer assay to measurethe amount (number) of CD8+ T-cells that recognize (and/or bind orselectively bind) a specific epitope in a recombinant polypeptide of theinvention.

[0742] Putative MHC class I binding sequences occurring in recombinantpolypeptides of the invention are identified by sequence analysis usinga suitable epitope-identification algorithm or program, such as theBONSAI algorithm developed at Stanford University, the TEPITOPEalgorithm, the SYFPEITHI program (which applies the algorithm ofRammensee et al.), the MAPPP program (available athttp://www.mpiib-berlin.mpg.de/MAPPP/), the PREDEP program (available athttp://bioinfo.md.huji.ac.il/marg/Teppred/mhc-bind/), the ProPredprogram (http://www.imtech.res.in/raghavalpropred), and the BIMASprogram (available at http://bimas.dcrt.nih.gov/molbio) which arevariously described in, e.g., Altuvia et al., Mol. Immunol., 31, 1-19(1994), Brusic et al., Nuc Acids Res., 22, 3663-3665 (1994), Hammer etal., J. Exp. Med., 180, 2353-2358 (1994), Parker et al., J. Immunol.,152, 163-175 (1994), Sturniolo et al. Adv. Immunol., 66, 67-100 (1997),and Cunha-Neto, Braz. J. Med. Biol. Res., 32(2), 199-205 (1999), orother suitable software to analyze the amino acid sequence ofrecombinant polypeptides of the invention. The amino acid sequences oftest polypeptides also can be subjected to sequence analysis identifypredicted T cell epitopes using software programs such as EpiPlot 1.0(available at http://genomics.com/software/files/epiplot1.exe), theEPIMER/EPIMAX algorithm developed at the Brown University School ofMedicine (currently managed by EpiVax, Inc. (Providence, R.I.), or asequence analysis algorithm such as the SOHHA algorithm (described inReyes et al., Methods Enzymol 202:225-38 (1991)). In addition,information and software programs for identifying and/or predicting Tcell epitopes using software programs in polypeptide sequences of theinvention can be obtained at the following websites:http://www.epitope-informatics.com, http://www.vaccinome.com/index.htm,http://syfpeithi.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm,http://www.imtech.res.in/raghava/propred/index.html,http://bimas.dcrt.nih.gov/molbio/hla_bind/http://epivax.com/epitope.html,and http://www.tepitope.com.

[0743] Related techniques for identifying T cell and B cell epitopes aredescribed in, e.g., International Patent Application WO 99/53058,Kammerer et al., Clin Exp Allergy 27:1015-1026 (1997), Sakakibara et al.J Vet Med Sci 60:599-605 (1998), Jiang et al., Protein Sci 9:403-416(2000), Milik et al., Nat Biotech 16(8):753-756 (1998), Walshet et al.,J Immunol Meth, 121:1275 (1989), and Schoofs et al. J Immunol140:611-616 (1987).

[0744] T cells are isolated and purified as described in Example 22. TheT cells are mixed with a polypeptide comprising a single predicted MHC Ibinding sequence or T cell epitope identified by, e.g., one of theabove-described methods/analyses, joined to a class I HLA molecule (amolecule on the surface of CD8+ cells that helps display the epitope tothe immune system)—more particularly, a synthetic tetrameric form of afluorescently labeled MHC Class I molecule. As CD8+ T cells recognizeantigen in the form of short peptides bound to MHC class I molecules, Tcells with the appropriate T cell receptor bind to the labeled tetramersand can be quantified by flow cytometry as described in Example 2. Tcells with receptors for a specific epitope without prior antigenstimulation are quantified. The results of such experiments are highlyquantitative and sensitive.

[0745] At least some of the recombinant polypeptides contain epitopesthat react with (or bind or specifically bind to) T cells in theabove-described assay. The results of these experiments confirm thecytotoxic effects attendant at least some of the recombinantpolypeptides of the invention.

[0746] This experiment provides a suitable assay for quantifying CD8+ Tcells that react with (or bind or specifically bind to) a polypeptide ofthe invention. This experiment also provides a technique for identifyingnovel T cell epitopes in such polypeptides.

Example 27

[0747] This example illustrates a method of immunization of rhesusmacaque monkeys with an effective amount of an immunogen-encoding orantigen-encoding nucleotide sequence of the invention sufficient toinduce a protective immune response(s) in the monkeys against one ormore (and preferably against two or more) dengue virus serotypes. Suchan amount may be an immunogenic amount or an antigenic amount. DNA-basedimmunization methodologies using wild-type dengue virus antigens andrhesus macaque monkeys have been described (see, e.g., the methodsdescribed in Raviprakash et al., J. Gen. Virology 81:1659-1667 (2000),which is incorporated herein by reference in its entirety for allpurposes), and such methods can alternatively be employed herein oradapted as desired.

[0748] RNA or DNA sequences of the invention can be used in the methodsdescribed below. In the studies below, an immunogen- or antigen-encodingDNA sequence is employed for each study. Each study is conductedaccording to the guidelines in “Guide for the Care and Use of LaboratoryAnimals, Institute of Laboratory Animal Resources,” National ResearchCouncil, DHHS Publication,No. NIH-86-23 (1985). Animals are obtainedfrom the Walter Reed Army Institute of Research animal facility inForest Glen, Md.

[0749] Rhesus macaque monkeys (Macaca mulatta), aged approximately 7 to24 years, are pre-tested for the presence of antibodies against dengueviruses of each of the four dengue virus serotypes by ELISA assay andplaque-reduction neutralization test. Those monkeys that do not showevidence of exposure to such dengue viruses (e.g., are sero-negative forsuch dengue viruses) are divided into several groups of threemonkeys/group. Each monkey in a non-control group is inoculatedintramuscularly (i.m.) or intradermally (i.d.) with a compositioncomprising an immunogenic amount or antigenic amount (e.g., about 0.1 μgto about 5 mg) of pMaxVax10.1 DNA plasmid vector comprising a nucleicacid sequence of the invention (as described in Example 22) in acarrier, such as, e.g., an endotoxin-free carrier, such as, e.g., apharmaceutical carrier (e.g., PBS or another suitable saline buffer,0.1M NaCl, or any suitable carrier which maintains the pH of thesolution at about 7.4); the carrier optionally comprises additionalexcipients such as preservative agents. Alternatively, another plasmidvector comprising such nucleotide sequence of the invention can be used.Each monkey of a particular group is immunized with the same vector inthe same concentration in the same manner.

[0750] In one study, a pMaxVax10.1 vector comprising a nucleic acidsequence encoding a recombinant PRM15/tE dengue virus antigen fusionprotein is tested. In such study, e.g., each monkey in a group isadministered with a pMaxVax10.1 vector comprising a nucleic acidsequence comprising a recombinant PRM15 gene and recombinant truncatedenvelope (E) gene. Such nucleic sequence comprises, e.g., one of thefollowing: SEQ ID NOS:157 (clone 2G11), 158 (clone 5/21), 159 (clone6E12), 185 (clone 16B4), 187 (clone 16G11), 200 (clone 18H2), 172 (clone18E11), and 235 (clone 18H6).

[0751] In a second study, a pMaxVax 10.1 vector comprises a nucleic acidsequence encoding a recombinant C15/full length prM/full length E denguevirus antigen fusion protein. In such study, e.g., each monkey in agroup is administered with a vector comprising a recombinant C15 gene,recombinant full length prM gene and recombinant full length E gene.Such nucleic sequence comprises, e.g., one of the following: SEQ IDNOS:254 (clone 16G11-D4), 255 (clone 16G11-25B10ext), 256 (clone18H6-D4), and 257 (clone 18H6-25B10ext).

[0752] As a control, each monkey of a control group of three monkeys ismock immunized with the carrier alone (with no plasmid vector). As asecond control, each monkey of another group of three test monkeys isimmunized with the same amount of pMaxVax10.1_(null) plasmid vectorcontrol (null vector) (e.g., for i.m. or i.d. injection, 1 mg ofpMaxVax10.1_(null) plasmid vector is administered in the same totalvolume, in one injection or divided in several injections, as inadministration of recombinant vectors).

[0753] While intramuscular and intradermal injection administrations areamong the preferred routes of administration, vectors comprising theimmunogen-encoding or antigen-encoding DNA or RNA sequences of theinvention (and vector compositions thereof) can be administered by avariety of standard routes of administration as described herein, vectorcompositions can be any of those described herein, including, e.g., butnot limited to, needle injection, parenteral administration,subcutaneous administration, gene gun or other biolistic deliverydevice, impression through the skin, oral delivery, inhalation, topicalor transdermal delivery (e.g., using a transdermal patch or ointment).

[0754] The total amount of recombinant plasmid vector administereddepends upon the manner of administration. For administration via needleinjection, about 0.3 mg to about 2 mg, usually about 1 mg, of such DNAplasmid vector is typically administered. For administration via genegun, about 0.1 μg to about 100 μg plasmid vector, typically from about 1μg to about 10 μg plasmid vector, are administered.

[0755] The total volume of the plasmid vector composition for eachimmunization typically depends upon the amount or dose of DNA vector (inmg) to be administered. For 1 mg DNA vector administered via injection,the total volume is typically about 0.5 ml. The total volume of thevector composition can be administered in one administration or dividedinto several smaller volumes administered in several administrationsconsecutively, sequentially, or simultaneously, at one or more sites inthe animal.

[0756] For example, for intramuscular administration, 0.5 ml totalvolume of DNA plasmid vector composition is typically administered to amonkey in one injection or two injections in a muscle of the monkey,such as, e.g., the tibialis anterior muscle. For intradermaladministration, e.g., 0.5 ml total volume of DNA plasmid vectorcomposition is typically administered to a monkey (e.g., anteriorthoracic dermal area of monkey) in five administrations of 0.1 ml each.One of skill in the art can employ and/or adapt other administrationformats, volumes, and compositions known in the art.

[0757] The DNA plasmid vector is optionally administered with one ormore transfection promoting agents (including, e.g., those describedherein), one or more immunostimulatory sequences (including, e.g.,nucleotide sequences comprising CpG islands or unmethylated CpG motifs,termed ISS motifs) (see Krieg, Trends in Microbiol 7:64-65 (1999)), oneor more cytokines (e.g., GM-CSF), one or more adjuvants (e.g., LAMP,alum, other known adjuvants), and/or one or more costimulatorymolecule-encoding sequences. Such costimulatory molecule-encodingsequences may include, but are not limited to, e.g., at least onemammalian B7-1-encoding nucleotide sequence or B7-1 homolog-encodingnucleotide sequence, at least one mammalian B7-2-encoding nucleotidesequence or B7-2 homolog-encoding nucleotide sequence, or at least onenovel co-stimulatory polypeptide-encoding nucleotide sequence describedin International Patent Application WO 02/00717, filed Jun. 22, 2001(including e.g., at least nucleotide sequence encoding, e.g., a CD28binding protein (“CD28BP”), such as CD28BP-15), or any combinationthereof. Adjuvants can be delivered to a subject with one or morerecombinant dengue polypeptides of the invention. In one format, a DNAvector comprising a sequence that encodes a recombinant antigenpolypeptide and an intracellular targeting adjuvant (e.g., LAMP proteinsequence) and/or lysosomal associated membrane protein can be used fordelivery to a subject.

[0758] The immunizations (e.g., vaccinations) are repeated at leasttwice after the initial inoculation with the identical vectorcomposition in the same concentration (e.g., 1 mg vector/0.5 ml totalvolume). Each subsequent inoculation or immunization is often termed a“boost.” The second inoculation (i.e., first “boost”) is administeredtwo weeks or one month after initial inoculation. The third inoculation(i.e., second “boost”) is typically administered at about 2 to about 6months following the initial inoculation (most typically at about 4,about 5, or about 6 months after the initial inoculation). Those monkeysreceiving intradermal inoculation(s) can be inoculated a fourth timewith the same vector in the same amount at 12, 14 or 18 months after theinitial inoculation. Groups of control animals are administered with an“empty” plasmid vector (e.g., pMaxVax10.1_(null)) in the sameconcentration (e.g., 1 mg/0.5 ml) or a carrier (e.g., pharmaceuticalcarrier) in the same volume (e.g., 0.5 ml) alone during the first(initial), second, third, and fourth inoculations.

[0759] Alternatively, for another group(s) of monkeys, one or morerecombinant polypeptides of the invention can be used for theinoculation or immunization boosts following the initial inoculation asdescribed in Example 28. The effectiveness of one or more boosts using apolypeptide antigen of the invention versus an immunogen-encoding orantigen-encoding DNA or RNA sequence of the invention is compared. Suchpolypeptide antigens can be administered by a variety of standard routesof administration as described herein, including, e.g., parenteral,intramuscular and intradermal delivery (see Example 28).

[0760] While intramuscular and intradermal injection administrations areamong the preferred routes of administration, vectors comprising theimmunogen-encoding or antigen-encoding DNA or RNA sequences of theinvention can also be administered by a variety of standard routes ofadministration as described herein, including, e.g., parenteraladministration, subcutaneous administration, or by biolistic injection(e.g., a gene gun).

[0761] Sera are obtained from each monkey (e.g., by bleeding each monkeydaily for 5 to 10 days). The sera are tested for the presence ofantibodies specific for each dengue virus serotype (DEN-1, DEN-2, DEN-3,and DEN-4) by ELISA assay and/or PRNT assay as described previously. Theability of sera of inoculated monkeys to neutralize each of the fourdengue virus serotypes is determined by PRNT assay. A negative controlfor the PRNT assays consists of a pool of the sera obtained from monkeysprior to being immunized A “prime” is typically defined as the firstimmunization.

[0762] The effectiveness of the prophylactic administration ofrecombinant polypeptide-encoding nucleotide acids of the invention insuch amount(s) is assessed by challenge of the immunized monkeys withone or more dengue viruses, preferably of multiple dengue virusserotypes, and observation of the occurrence of viremia (including,e.g., onset, levels, and duration of viremia) or nonoccurrence ofviremia in such monkeys thereafter.

[0763] Groups of monkeys are selected for in vivo challenge with one ormore live dengue viruses of the four serotypes. For one challenge study,monkeys are challenged with a dengue virus at about 9 or at about 12months after the initial immunization. For a second challenge study,monkeys are challenged with a dengue virus at about 15 or at about 18months after the initial immunization. For each challenge study, acontrol group of monkeys is also maintained and observed for comparison.Serum is obtained by bleeding from each monkey prior to the viralchallenge. Then, each monkey is inoculated via, e.g., subcutaneousinjection in a leg or arm with about 0.5 ml of a solution comprisingabout 1×10² to about 5×10⁴ plaque forming units (p.f.u.) (typicallyabout 1×10² to about 1.5×10⁴ p.f.u., 1×10² to about 1.25×10⁴ p.f.u.,about 1×10³ p.f.u. or about 1.25×10⁴ p.f.u.) of a live dengue virus ofone of the four serotypes. At least two monkeys are injected with adengue virus of one serotype; all dengue virus serotypes are employed ineach challenge study.

[0764] Sera are obtained by bleeding the inoculated and control monkeysat about 15 and about 30 days after initial challenge; alternatively,sera can be obtained at other days following initial challenge. Viremiaand antibodies specific for each dengue virus serotype are measured inthe sera. Viremia is measured by following the procedure set forth inRaviprakash et al., J. Gen. Virology 81:1659-1667 (2000). Antibodyresponse is measured as described previously or by methods known in theart (see, e.g., Raviprakash et al., J. Gen. Virology 81:1659-1667(2000)). Responses in monkeys immunized with a plasmid vector encoding arecombinant antigen of the invention are compared with monkeys injectedwith the null vector or with carrier alone.

[0765] For those monkeys inoculated with a plasmid vector comprising arecombinant dengue virus antigen-encoding nucleic acid of the inventionthat survive challenge with a particular dengue virus, such recombinantdengue virus antigen-encoding nucleic acid induces a protective immuneresponse against infection by such dengue virus. Protection may be fullor partial protection. Full protection is observed in those immunizedmonkeys that are completely protected from developing viremia; partialprotection is observed in those immunized monkeys that only develop areduced viremia compared to control monkeys.

[0766] The present invention includes recombinant nucleic acids thatencode recombinant PRM15/tE dengue virus antigen fusion proteins and/orrecombinant C15/full length prM/full length E dengue virus antigenfusion proteins (and vectors thereof) that induce protective immuneresponse(s) in vivo against dengue virus infection by at least onedengue virus serotype, preferably against at least two dengue virusserotypes, more preferably against at least three or at least fourdengue virus serotypes, following in vivo challenge by one or moredengue virus serotypes in a non-human primate model, e.g., rhesusmacaque monkey model.

[0767] In another aspect, the invention includes recombinant nucleicacids that encode recombinant tE dengue virus antigen fusion proteinsand/or recombinant full length prM/full length E dengue virus antigenfusion proteins (and vectors thereof) that induce protective immuneresponse(s) in vivo against dengue virus infection by at least onedengue virus serotype, preferably against at least two dengue virusserotypes, more preferably against at least three or at least fourdengue virus serotypes, following in vivo challenge by one or moredengue virus serotypes, in a non-human primate model. In such aspect,the encoded recombinant polypeptide does not include the PRM15 signalpeptide or C15 signal peptide.

[0768] The invention also includes neutralizing antibodies that protectagainst dengue virus infection by one of the four dengue virusserotypes. Such antibodies are produced in response to administration toa subject of a recombinant nucleic acid that encodes a recombinantPRM15/tE dengue virus antigen fusion protein or a recombinant C15/fulllength prM/full length E dengue virus antigen fusion protein and can beisolated by standard methods from the sera of such subjects. Vectorscomprising such nucleic acids may be administered to the subject.

Example 28

[0769] This example illustrates a method of immunization of rhesusmacaque monkeys with an effective amount of one or more recombinantdengue virus polypeptide antigens of the invention sufficient to inducea protective immune response(s) in the monkeys against one or more (andpreferably against two or more) dengue viruses. Such an amount may be animmunogenic amount or an antigenic amount.

[0770] Immunization methodologies using dengue virus antigens andprimates have been described, and such methods can alternatively beemployed herein or adapted as desired. See, e.g., Eckels et al., Amer JMedicine and Hygiene (1994) 50:472-487; Raviprakash et al, J. Gen.Virol. (2000) 81: 1659-1667).

[0771] A recombinant polypeptide of the invention can be used in themethods described below. Each study is conducted according to theguidelines in “Guide for the Care and Use of Laboratory Animals,Institute of Laboratory Animal Resources,” National Research Council,DHHS Publication No. NIH-86-23 (1985). Animals are obtained from theWalter Reed Army Institute of Research animal facility in Forest Glen,Md.

[0772] Rhesus macaque monkeys (Macaca mulatta), aged approximately 7 to24 years, are pre-tested for the presence of antibodies against dengueviruses of each of the four dengue virus serotypes by ELISA assay andplaque-reduction neutralization test. Those monkeys that do not showevidence of exposure to such dengue viruses (e.g., are sero-negative forsuch dengue viruses) are divided into several groups of threemonkeys/group. Each monkey in a non-control group is inoculatedintramuscularly (i.m.) or intradermally (i.d.) on day 0 with acomposition comprising an immunogenic amount or antigenic amount (e.g.,about 0.1 μg-1 mg, about 0.5-500 μg, about 0.5-100 μg, about 1-50 μg,about 1-10 μg, about 5-30 μg, about 15-25 μg, or about 0.5 μg to about 2μg) of a recombinant polypeptide of the invention in a carrier (asdescribed in Example 22). The carrier may comprise, e.g., anendotoxin-free carrier, such as, e.g., a pharmaceutical carrier (e.g.,PBS or another suitable saline buffer, 0.1M NaCl, or any suitablecarrier which maintains the pH of the solution at about 7.4) and mayoptionally comprise additional excipients such as preservative agents.The recombinant polypeptide may be administered with an adjuvant (e.g.,Freund's incomplete adjuvant (FIA) or aluminum hydroxide) and/orliposome(s) in an amount such as is typically delivered with a DNAvector or DNA vaccine. One of skill can determine the type of adjuvantor liposome and the amount to be included. One group of monkeys servesas the control groups; monkeys of the control group are mock immunizedwith PBS or another suitable saline solution.

[0773] Alternatively, a monkey is administered with a compositioncomprising a carrier (e.g., pharmaceutically acceptable carrier) and anucleic acid vector encoding a recombinant polypeptide of the invention.A sufficient amount of the composition is administered such that animmunogenic amount or antigenic amount of the encoded polypeptide isproduced (e.g., from about 50 μg to about 10 mg, about 100 μg to about 5mg, about 1-5 mg, or from about 2-5 mg nucleic acid is administered).Typically, the carrier is PBS. At least one adjuvants and/or liposomemay be included in the composition in an amount as is typicallydelivered with a DNA vector or DNA vaccine. One of skill could readilydetermine the type of adjuvant or liposome and the amount to beincluded.

[0774] In one study, a group(s) of monkeys is administered with at leastone recombinant dengue virus polypeptide of the invention comprising aPRM15/tE dengue virus antigen fusion protein (or combination of suchpolypeptides). For example, such polypeptide(s) may comprise one or moreof the following sequences: SEQ ID NOS:66 (clone 2G11), 67 (clone 5/21),69 (clone 6E12), 89 (clone 16B4), 93 (clone 16G11), 109 (clone 18H2),108 (clone 18E11), and 110 (clone 18H6). Alternatively, the recombinantpolypeptide that is administered comprises a recombinant tE dengue viruspolypeptide antigen and does not include a PRM15 signal peptide.

[0775] In a second study, a group(s) of monkeys is administered with atleast one recombinant dengue virus polypeptide of the inventioncomprising a recombinant C15 signal peptide/full length prM/full lengthE dengue virus antigen fusion protein. Such polypeptide comprises, e.g.,one of the following sequences: SEQ ID NOS:250 (clone 16G11-D4), 251(clone 16G11-25B10ext), 252 (clone 18H6-D4), and 253 (clone18H6-25B10ext). Alternatively, the recombinant polypeptide that isadministered comprises a recombinant full length prM/full length Edengue virus antigen fusion protein and does not include a C15 signalpeptide. As will be understood by those of skill, other polypeptides ofthe invention may be employed in these methods. Preferably, arecombinant tetravalent dengue virus polypeptide antigen of theinvention is employed.

[0776] As described in Example 27, such recombinant dengue viruspolypeptide can be optionally be administered with one or moretransfection promoting agents (including, e.g., known agents and thosedescribed herein), one or more immunostimulatory sequences (e.g.,nucleotide sequences comprising CpG islands or unmethylated CpG motifs,ISS motifs), one or more cytokines (e.g., GM-CSF), one or moreadjuvants, and/or one or more costimulatory molecule polypeptides (e.g.,B7-1 polypeptide, B7-2 polypeptide, a novel co-stimulatory polypeptidedescribed in International Patent Application WO 02/00717, filed Jun.22, 2001 (including e.g., CD28 binding protein, such as CD28BP-15), or acombination thereof, as well as a carrier(s) or excipient(s), such as,e.g., a preservative agent(s).

[0777] The manner of administration of the polypeptide compositions canbe any of those described herein, including, e.g., but not limited to,needle injection, impression through the skin, subcutaneousadministration, parenteral administration, oral delivery, inhalation, ortopical or transdermal delivery (e.g., using a transdermal patch orointment). The total amount of recombinant polypeptide administereddepends upon the manner of administration. For administration via needleinjection, about 0.1 μg-1 mg, about 0.5-500 μg, about 0.5-100 μg, about1-50 μg, about 1-10 μg, about 5-30 μg, about 15-25 μg, or about 0.5 μgto about 2 μg of such recombinant polypeptide can be administered. Oneof skill in the art can readily determine the amount of polypeptidepreferable for other routes of administration.

[0778] The total volume of the polypeptide composition for eachimmunization typically depends upon the amount or dose of polypeptide(in mg) to be administered as described in Example 27. The total volumeof the composition can be administered in one administration or dividedinto several smaller volumes administered in several administrationsconsecutively, sequentially, or simultaneously, at one or more sites inthe animal. For example, for intramuscular administration, the totalvolume of the polypeptide composition may be administered to a monkey inone injection or two injections in a muscle of the monkey, such as,e.g., the tibialis anterior muscle. For intradermal administration, asimilar total volume of polypeptide composition can be divided intosmaller, equal volumes and delivered to a monkey (e.g., anteriorthoracic dermal area of monkey) in multiple administrations. One ofskill in the art can employ and/or adapt other administration formats,volumes, and compositions known in the art.

[0779] The immunizations (e.g., vaccinations) are repeated at leastabout two times after the initial inoculation with identical polypeptidein the same amount and concentration. Each subsequent inoculation orimmunization with the polypeptide is termed a “boost.” The secondinoculation or immunization (e.g., the first “boost” following theinitial inoculation) is administered about 2 weeks to 1 month after theinitial inoculation, and a third inoculation (e.g., the second “boost”)is administered about 1 to about 6 months following the secondinoculation (most typically from about 3 months, 4 months, or about 6months). Control animals are administered with a carrier (e.g.,pharmaceutical carrier) alone in the same volume during the second andthird inoculations following the initial inoculation.

[0780] Alternatively, for another group(s) of monkeys, one or more DNAor RNA sequences of the invention (as described above) can be used forone or more of the inoculation boosts following the initial inoculation,as described in Example 27. The effectiveness of boosts using apolypeptide antigen of the invention versus an immunogen-encoding orantigen-encoding DNA or RNA sequence of the invention is compared.

[0781] Following the procedures set forth in Example 27, sera areobtained from each monkey, and such sera are tested for the presence ofantibodies specific for each dengue virus serotype (DEN-1, DEN-2, DEN-3,and DEN-4) by ELISA assay and/or PRNT assay. The ability of sera ofinoculated monkeys to neutralize each of the four dengue virus serotypesis determined by PRNT assay.

[0782] Effectiveness of the prophylactic administration of suchrecombinant polypeptide(s) of the invention is assessed by challenge ofthe immunized monkeys with one or more dengue viruses, preferably ofmultiple dengue virus serotypes, and observation of the occurrence ofviremia (including, e.g., onset, levels, and duration of viremia) ornonoccurrence of viremia in such monkeys thereafter, as described inExample 27.

[0783] The administration of an effective amount (e.g., immunogenicamount or antigenic amount) of one or more recombinant polypeptides ofthe invention to a nonhuman primate induces a protective immune responseagainst in vivo challenge by at least one dengue virus. Protection maybe full or partial protection. Full protection is observed in thoseimmunized monkeys that are completely protected from developing viremia;partial protection is observed in those immunized monkeys that onlydevelop a reduced viremia compared to control monkeys.

[0784] The present invention includes recombinant PRM15/tE dengue virusantigen fusion proteins and recombinant C15/full length prM/full lengthE dengue virus antigen fusion proteins (and compositions thereof) thatinduce protective immune response(s) in vivo against dengue virusinfection by at least one dengue virus serotype, preferably at least twodengue virus serotypes, more preferably at least three or at least fourdengue virus serotypes, following in vivo viral challenge by one or moredengue virus serotypes, in a non-human primate model, e.g., rhesusmacaque monkey model.

[0785] In another aspect, the invention also includes recombinanttruncated E dengue virus antigen fusion proteins and recombinant fulllength prM/full length E dengue virus antigen fusion proteins (andcompositions thereof) that induce protective response(s) in vivo againstdengue virus infection by at least one dengue virus serotype, preferablyat least two dengue virus serotypes, more preferably at least three orat least four dengue virus serotypes, following in vivo viral challengeby one or more dengue virus serotypes, in a non-human primate model. Insuch aspect, the encoded recombinant polypeptide does not include thePRM15 signal peptide or C15 signal peptide.

Example 29

[0786] This example illustrates a method of immunization of a human witha composition comprising a pharmaceutically acceptable carrier (e.g.,PBS) and at least one recombinant immunogen-encoding or antigen-encodingDNA or RNA sequence of the invention or with at least one human codonoptimized DNA or RNA sequence of the invention described herein. Forexample, any recombinant PRM15/tE fusion protein-encoding nucleotidesequence, including, e.g., but not limited to, any of SEQ ID NOS:157(clone 2G11), 158 (clone 5/21), 159 (clone 6E12), 185 (clone 16B4), 187(clone 16G11), 200 (clone 18H2), 172 (clone 18E11), 235 (clone 18H6), orany composition or mixture of such nucleotide sequences, or anyrecombinant C15/full length prM/full length E fusion protein-encodingnucleotide sequence, including, e.g., but not limited to, any of SEQ IDNOS:254 (clone 16G11-D4), 255 (clone 16G11-25B10ext), 256 (clone18H6-D4), 257 (clone 18H6-25B10ext), 204 (2G11-D4), and 202 (6E12-D4),or any mixture of such nucleotide sequences, can be employed in thismethod. In another aspect, a composition comprising a pharmaceuticallyacceptable carrier and at least one recombinant PRM15/tE fusionprotein-encoding nucleotide sequence and at least one recombinantC15/full length prM/full length E fusion protein-encoding nucleotidesequence can be employed (e.g., 18H6 (SEQ ID NO:235), 2G11-D4 (SEQ IDNO:204), and 6E12-D4 (SEQ ID NO:202). Other immune-stimulatingpolynucleotides of the invention or combinations of such polynucleotidescan also be used in this method.

[0787] Immunization of a human with one or more recombinant DNA or RNAsequences of the invention is done using a method similar to thatdescribed in Example 27. Prophylactic administration of an effectiveamount (e.g., immunogenic amount or antigenic amount) of one or moresuch DNA or RNA sequences of the invention to a human results in theinduction or enhancement of an immune response against at least one, andpreferably at least two, at least three, or at least four, dengueviruses in the human. For such polynucleotides of the invention, aprotective immune response against one or more dengue viruses is inducedor enhanced in the human recipient. Such response may be a partiallyprotective response, but is preferably a fully protective response. Suchpolynucleotide or combination of several different recombinantpolynucleotides serves as a DNA vaccine that induces an immuneresponse(s) that protects against infection by at least one, preferablyat least two, and more preferably at least three or four dengue virusserotypes.

[0788] In addition, the invention provides methods of prophylacticimmunization of humans with one or more recombinant dengue viruspolypeptides of the invention in, e.g., a suitable carrier, such as apharmaceutically acceptable carrier (PBS), and optionally any acceptableadjuvant, such as alum. Immunization of a human with one or morerecombinant polypeptides of the invention is conducted using a methodsimilar to that described in Example 27. Any recombinant polypeptide ofthe invention or combination of such polypeptides can be used for humanimmunization; for example, such polypeptide may comprise one or more ofthe following sequences: SEQ ID NOS:66 (clone 2G11), 67 (clone 5/21),69(clone 6E12), 89 (clone 16B4), 93 (clone 16G11), 109 (clone 18H2), 108(clone 18E11),and 110 (clone 18H6) or 251 (16G11-25B10). Alternatively,the recombinant polypeptide that is administered comprises a recombinanttE dengue virus polypeptide antigen and does not include a PRM15 signalpeptide. In another aspect, a combination of two or more any of therecombinant polypeptides of the invention can be administered.

[0789] Such polypeptides can be administered to a human in addition to apolynucleotide or, alternatively, in place of such polynucleotide. Therecombinant polypeptide is administered to the human in an amounteffective to induce or enhance an immune response(s) in the human. Theeffective amount comprises, e.g., immunogenic amount or antigenicamount. Suggested doses were described above. Such antigenicpolypeptides may serve as protein vaccines. Administration of thepolypeptide can be via a variety of routes, as described above;transdermal and intramuscular administration via injection are among thetypical routes of polypeptide delivery.

[0790] Amounts of recombinant dengue virus polynucleotides andpolypeptides used for the immunization of rhesus macaque monkeys inExamples 27 and 28 can be similarly used for the immunization of humans;alternatively, such amounts can be modified proportionally to take intoaccount the size of the human relative to that of the macaque monkey.For example, the dose of dengue virus peptide antigen for immunizationof a human may be slightly higher (e.g., about 0.5 μg-1 mg, about0.5-500 μg, about 0.5-100 μg, about 1-50 μg, about 1-10 μg, about 5-30μg, about 15-25 μg) compared to the immunization dose used for a monkey.The prophylactic administration of an effective amount of one or morerecombinant dengue virus polypeptide antigens of the invention inducesin a human a protective immune response against at least one, preferablyat least two, and more preferably at least three or at least four,dengue viruses.

[0791] Such polypeptides and polynucleotides of the invention can beformulated as a composition with a carrier or excipient, including as apharmaceutical composition, as described herein, and can be administeredto a human by a variety of routes as described herein (see also Examples27 and 28). Such polynucleotides can be delivered via a vector asdescribed herein (see, e.g., Example 27). The recombinant dengue viruspolypeptide of the invention can be administered alone (e.g., as aprotein vaccine) or in combination (before or after) with administrationof a plasmid vector comprising the corresponding polypeptide-encodingpolynucleotide of the invention. Other formats are set forth in Example28.

[0792] Similarly, the polynucleotide can be administered alone via aplasmid vector (e.g., as a DNA vaccine) or in combination (before orafter) with administration of the corresponding encoded polypeptide ofthe invention, and a variety of boosting strategies and methodologiescan also be employed, as described in, e.g., in Examples 27 and 28. Forexample, at least one such recombinant polynucleotide (e.g., recombinantDNA vaccine) or at least such recombinant polypeptide (e.g., recombinantprotein vaccine) can be administered to a human initially in an amounteffective to induce an immune response, and after an appropriate timeperiod (which can be determined by one of skill in the art) (e.g., aboutone week, about one month, about 4, about 5, about 6, about 12, or about18 months, etc.), a “boost” immunization comprising the same or adifferent amount of the polynucleotide (DNA vaccine) or polypeptide(protein vaccine) can be administered to the same human. If desired,multiple boosts can be administered to the human.

[0793] In another aspect, the invention provides methods comprising theadministration to a human of an effective amount of a compositioncomprising at least one nucleic acid encoding at least one dengue virusantigen of the invention in combination with one or more nucleic acidsencoding one or more WT dengue virus prM/E fusion proteins, WT denguevirus PRM15/tE fusion proteins, and/or WT C15/full length prM/fulllength E fusion proteins, and, optionally, a nucleotide encoding aco-stimulatory molecule or cytokine, such as GM-CSF or an interferon, orcombination of such co-stimulatory molecule-encoding nucleotides and/orcytokine-encoding nucleotides, wherein the amount is sufficient toinduce a protective immune response against at least one, at least two,and preferably at least three or at least four dengue virus serotypes.

[0794] In yet another aspect, the invention provides methods comprisingthe administration to a human of an effective amount of a compositioncomprising at least one recombinant dengue virus antigen of theinvention in combination with one or more WT dengue virus prM/E fusionproteins, WT dengue virus PRM15/tE fusion proteins, and/or WT C15/fulllength prM/full length E fusion proteins, and, optionally, one or moreco-stimulatory molecules or cytokines, such as GM-CSF or an interferon,or combination of such co-stimulatory molecules and/or cytokines,wherein the amount is sufficient to induce a protective immune responseagainst at least one, at least two, and preferably at least three or atleast four dengue virus serotypes.

[0795] In all such methods and formats described herein, the resultinginduced immune response(s) induced in the human who received the one ormore immunizations of a polynucleotide(s) and/or polypeptide(s) can bedetermined at various times, including before and/or after the variousimmunizations, by measuring, e.g., the antibody titer level in serum(blood) obtained from the human using ELISA assays and/or PRNT assaysdescribed above.

[0796] The sequence listing is shown below. SEQ NO. NAME SEQUENCE 1.2/7-NPRM MRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDTELIKTEATQPATLRK“NPRM”= YCIEAKLTNTTTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITC noprM AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQ seqLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 2. 2G11-NPRMMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLET (no prMYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 3. 5/21-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC seq)AKFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGESYIVVGVGDSALTLHWFRKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGA 4. 6C6-NPRMMRCVGIGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRT (no prMYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGIVTC seq)AMFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMILMKMKNKAWMVHRQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSAYT 5. 6E12-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDTELLKTEVTNPAVLRK (no prMLCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGTTTIFAGHLKCKVRMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFG 6. 6F4-NPRMMRCVGIGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDFELIKTEATQPATLRK (no prMYCIEAKLTNTTTESRCPTQGEAILPEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFEATARGARRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 7. 7A9-NPRMMRCVGIGNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRK (no prMLCIEASISNITTATRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLVTC seq)AKFQCLEPIEGKVVQHENLKYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMILLTMKSKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKNAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 8. 11B1-NPRMMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELIKTEATQPATLRK (no prMYCIEAKLTNTTTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHTALTGATEVDSGDGNHMFAGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITANPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFRKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGAIYGA 9. 11BB-NRPMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKLTNTTTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNHMFAGHLKCKVRMEKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGQMFEATARGARRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFG 10. 11C4-NPRMMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRT (no prMYCIEASISNITTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMILLTMKSKTWLVHKQWFLDLPLPWTAGADTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVGDKALKLHWFKKGSSIGKMFEATARGAKRMAILGETAWDFGSAYT 11. 11C11-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRK (no prMLCIEASISNITTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKTWLVHKQWFLDLPLPWTAGADTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGEDAPCKIPFSTEDGQGKAHNGRLITANPIVTEKDSPVNIEAEPPFGDSYIVVGAGEKALKLHWFKKGSSIGQMFEATARGARRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 12. 11E2-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRK (no prMLCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC seq)AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKRQDVTVLGSQEGAMHSALTGATEIQMSSGNLLFAGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGESYIVVGVGDSALTLHWFRKGSSIGQMFEATARGAKRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 13. 11E3-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELIKTTAKEVALLRT (no prMYCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC seq)AKFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGESYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 14. 12H4-NPRMMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELIKTEATQPATLRK (no prMYCIEASISNITTDSRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFEATARGARRMAILGDTAWDFGSAYT 15. 13E2-NPRMMRCVGIGNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRT (no prMYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGTDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGKMFEATARGAKRMAILGETAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFG 16. 13E11-NPRMMRCVGVGNRDFVEGVSGGAWVDVVLEHGGCVTTMAKNKPTLDIELIKTEATQPATLRK (no prMYCIEASISNITTDTRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGTVTMECSPRTGLDFNEMILLTMKSKTWLVHKQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGTDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGESYIVVGAGEKALTLHWFRKGSSIGQMFETTMRGAKRMAILGETAWDFGSVGGVFTSIGKALHWVFGAIYGA 17. 13F11-NPRMMRCVGIGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDIELIKTEATQPATLRK (no prMYCIEAKITNITTDSRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDEKGKAHNGRLITANPIVIDKEKPVNIELEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFESTYRGAKRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 18. 14B1-NPRMMRCVGISNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKITNITTDSRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGNIVQPENLKYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKRQDVTVLGSQEGAMHSALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFESTARTARRMAILGDTAWDFGSAYT 19. 14E9-NPRMMRCVGIGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDIELIKTEATQPATLRK (no prMYCIEAKITNITTDSRCPTQGEAILPEEQDQQYICRRDVVDRGWGNGCGLFGKGSVVTC seq)AKFQCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILMKMKNKAWMVHKQWFLDLPLPWTSGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSAYT 20. 14G10-NPRMMRCIGISNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKITNITTDTRCPTQGEAILPEEQDQQYICRRDVVDRGWGNGCGLFGKGSLVTC seq)AKFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTKFVPHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 21. 14H2-NPRMMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRT (no prMYCIEAKITNITTATRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITC seq)AKFQCVTKLEGNIVQPENLEYTIVITPHSGEEHAVGNDTQGVTVEITPQASTVEAILTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYKGEDAPCKIPFSSQDGQGKAHNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFETTMRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 22. 15C2-NPRMMRCVGISNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKITNITTDSRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKIVQYENLKYSVIVTVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCRLKMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGESYIVIGVEPGQLKLHWFKKGSSIGQMFEATARGARRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 23. 15D4-NPRMMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCLEPIEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGA 24. 15H4-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFELIKTEVTNPATLRKYCIEASISNITTATRCPTQGEANLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGRLKCRLKMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFESTYRGAKRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSVYTTMFG 25. 16B4-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEASISNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGTTTIFAGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGVGDKALKINWYKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFG 26. 16E8-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWFLDLPLPWTSGATTETPTWNRKELLVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFETTMRGAKRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 27. 16E10-NPRMMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKISNTTTDSRCPTQGEATLVEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGKIVQYENLKYSVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGALGLECSPRTGLDFNEMILLTMKNKAWMVHGQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGESYIVVGVGDSALTLHWFRKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSVYTTMFG 28. 16F12-NPRMMRCVGVGNGDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFELIKTIAKEVALLRTYCIEASISNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGTMHTALTGATEIQMSSGTTTIFAGHLKCKVKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPLEIMDLEKRHVLGRLITVNPTVIDKEKPVNIEAEPPFGESYIVVGVGDSALKINWYKKGSSIGKMFESTYRGARKMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 29. 16G11-NPRMMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEASISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSLGGLLTSLGKAVHQVFGSVYTTMFG 30. 17A12-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFELIKTEATQPATLRK (no prMYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITC seq)AKFKCLEPIEGKVVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTSGATTETPTWNRKELLVTFKNAHAKRQDVTVLGSQEGAMHSALTGATEIQTSGTTTIFAGHLKCRVRMDKLQLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPVSSQDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGARKMAILGDTAWDLGSVGGVFTSIGKALHQVFGAIYGA 31. 17D5-NPRMMRCIGISNRDFVEGVSGATWVDVVLEHGGCVTTMARNKPTLDIELIKTEATQPATLRK (no prMYCIEASISNITTATRCPTQGEAILPEEQDQQYICRRDVVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGA 32. 17D11-NPRMMRCVGTGNRDFVEGLSGATWVDVVLEHGGCVTTMAQGKPTLDIELIKTEATQPATLRK (no prMYCIEAKLTNTTTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 33. 17F5-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGVGDSALTLHWFRKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSLGKAVHQIFGSVYTTMFG 34. 17F11-NPRMMRCIGISNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRT (no prMYCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWLLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 35. 17G5-NPRMMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRT (no prMYCIEASISNITTATRCPTQGEAILKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQEVVVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMDKLTLKGVSYVMCTGSFKLEKETAETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 36. 17H3-NPRMMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKITNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKTWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKRQDVTVLGSQEGAMHSALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPAVTDKEKPVNIELEPPFGDSYIVVGAGDKALKLSWFKKGSSIGQMFESTYRGAKRMAILGETAWDLGSIGGVFTSVGK 37. 17H10-NPRMMRCIGISNRDFVEGVSGGSWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKITNITTDSRCPTQGEAIMPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC seq)AKFTCKKNMEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGVLTSLGKMVHQIFGSVYTTMFG 38. 17H12-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAQGKPTLDIELLKTEVTNPATLRK (no prMYCIEAKLTNTTTESRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFG 39. 18A9-NPRMMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKITNITTATRCPTQGEAILPEEQDQNYVCKHSMVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLTLKGVSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALTLHWFRKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVFTSVGK 40. 18B7-NPRMMRCIGISNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFELIKTTAKEVALLRT (no prMYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEALIPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 41. 18D7-NPRMMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRK (no prMLCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGTTTIFAGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTILIKVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 42. 18E9-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTEATQPATLRK (no prMYCIEAKLTNTTTATRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLPGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYSVIVTVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRKELLVTFKNAHAKRQDVTVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSVYTTMFG 43. 18E10-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRK (no prMYCIEAKLTNITTESRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTKFTAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 44. 18E11-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTVKEVALLRT (no prMYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITC seq)AKFKCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGKAHNGRLITANPIVIDKEKPVNIEAEPPFGESYIVVGAGDKALKLSWFKKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 45. 18H2-NPRMMRCVGVGNRDFVEGVSGGAWVDLVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRK (no prMYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGQGNGCGLFGKGSVVTC seq)AKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTSNHGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKKQDVVVLGSQEGAMHTALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFG 46. 18H6-NPRMMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRT (no prMYCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC seq)AKFTCKKNMEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKNAHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTEDGQGKAHNGRLITVNPIVIDKEKPVNIELEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGAIYGA 47. 21C1MRCIGISNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELIKTTAKEVALLRT ETRUNCYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTC only (noAKFKCVTKLEGKIVQYENLEYTVVVTVHNGDTHAVGNDTSNHGVTATITPRSPSVEVK prM)LPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMDKLQLKGMSYSMCTGKFQLVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGDSYIIIGVGDSALTLHWFRKGSSIGKMFESTYRGARRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 48. 23C12MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRK ETRUNLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC only (noAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILP prM) CEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNQHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFRKGSSIGQMFETTMRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 49. 23F5MRCVGIGNRDFVEGLSGGAWVDLVLEHGGCVTTMAKNKPTLDFELIKTEATQPATLRK ETRUNCYCIEAKLTNTTTESRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC only (noAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILP prM)EYGTLGLECSPRTGLDFNEMILLTMKNKTWLVHKQWFLDLPLPWTAGADTREVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCRLKMDKLELKGMSYSMCTGFKRIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGK 50. SEQUENCE M X₁ X₂ X₃ F I L X₄ M L V X₅ P SX₆ X₇ M R C V G X₈ G N R D F PATTERN 1 V E G X₉ S G X₁₀ X₁₁ W V D X₁₂ VL E H G X₁₃ C V T T M A K N K P T L D X₁₄ E L X₁₅ K T X₁₆ X₁₇ X₁₈ X₁₉X₂₀ X₂₁ L R X₂₂ X₂₃ C I E A X₂₄ I X₂₅ N X₂₆ T T X₂₇ X₂₈ R C P T Q G EX₂₉ X₃₀ L X₃₁ E E Q D X₃₂ X₃₃ X₃₄ X₃₅ C X₃₆ X₃₇ X₃₈ X₃₉ V D R G W G N GC G L F G K G S X₄₀ X₄₁ T C A K F X₄₂ C X₄₃ X₄₄ X₄₅ X₄₆ E G X₄₇ X₄₈ V QX₄₉ E N L X₅₀ Y T X₅₁ X₅₂ I T X₅₃ H X₅₄ G X₅₅ X₅₆ H X₅₇ V G N D T X₅₈X₅₉ X₆₀ G X₆₁ X₆₂ X₆₃ X₆₄ I T P Q X₆₅ S X₆₆ X₆₇ E A X₆₈ L X₆₉ X₇₀ Y G TX₇₁ X₇₂ X₇₃ E C S P R T G L D F N X₇₄ X₇₅ X₇₆ L L T M K X₇₇ K X₇₈ W X₇₉V H X₈₀ Q W F X₈₁ D L P L P W T X₈₂ G A X₈₃ T X₈₄ X₈₅ X₈₆ X₈₇ W N X₈₈ KE X₈₉ X₉₀ V T F K X₉₁ X₉₂ H A K X₉₃ Q X₉₄ V X₉₅ V L G S Q E G A M H X₉₆A L X₉₇ G A T E X₉₈ X₉₉ X₁₀₀ X₁₀₁ X₁₀₂ G X₁₀₃ X₁₀₄ X₁₀₅ X₁₀₆ F X₁₀₇ G HL K C X₁₀₈ X₁₀₉ X₁₁₀ M D K L X₁₁₁ L K G X₁₁₂ S Y X₁₁₃ M C T G X₁₁₄ FX₁₁₅ X₁₁₆ X₁₁₇ K E X₁₁₈ AN E T Q H G T X₁₁₉ X₁₂₀ X₁₂₁ X₁₂₂ V X₁₂₃ Y X₁₂₄G X₁₂₅ X₁₂₆ X₁₂₇ P C K I P X₁₂₈ X₁₂₉ X₁₃₀ X₁₃₁ D X₁₃₂ X₁₃₃ X₁₃₄ X₁₃₅X₁₃₆ X₁₃₇ X₁₃₈ G R L I T X₁₃₉ N P X₁₄₀ V X₁₄₁ X₁₄₂ K X₁₄₃ X₁₄₄ P V N I EX₁₄₅ E P P F G X₁₄₆ S X₁₄₇ I X₁₄₈ X₁₄₉ G X₁₅₀ X₁₅₁ X₁₅₂ X₁₅₃ X₁₅₄ L X₁₅₅X₁₅₆ X₁₅₇ W X₁₅₈ X₁₅₉ K G S S I G X₁₆₀ M F E X₁₆₁ T X₁₆₂ R G A X₁₆₃ R MAN I L G X₁₆₄ T A W D F G S X₁₆₅ G G X₁₆₆ X₁₆₇ T S X₁₆₈ G K X₁₆₉ X₁₇₀ HQ X₁₇₁ F G X₁₇₂ X₁₇₃ Y X₁₇₄ X₁₇₅ X₁₇₆ X₁₇₇ X₁₇₈ wherein X₁ is a V, T, orG residue, X₂ is a V or I residue, X₃ is an I or F residue, X₄ is a L orM residue, X₅ is an A or T residue, X₆ is a is a Y or M residue, X₇ isan A, T, or G residue, X₈ is a V or I residue, X₉ is a L or V residue,X₁₀ is an A or G residue, X₁₁ is an A or T residue, X₁₂ is a L or Vresidue, X₁₃ is a S or G residue, X₁₄ is an I or F residue, X₁₅ is a Lor I residue, X₁₆ is a T or E residue, X₁₇ is an A or V residue, X₁₈ isa K or T residue, X₁₉ is an E or N residue, X₂₀ is a V or P residue, X₂₁is a L, V, or T residue, X₂₂ is a K or T residue, X₂₃ is a L or Yresidue, X₂₄ is a K or S residue, X₂₅ is a T or S residue, X₂₆ is an Ior T residue, X₂₇ is an A or D residue, X₂₈ is a T or S residue, X₂₉ isan A or P residue, X₃₀ is an I, T, or Y residue, X₃₁ is a K or V Presidue, X₃₂ is a T or Q residue, X₃₃ is a Q or N residue, X₃₄ is a F orY residue, X₃₅ is a V or I residue, X₃₆ is a K or R residue, X₃₇ is a Hor R residue, X₃₈ is a T or D residue, X₃₉ is a V or F Y residue, X₄₀ isa L or V residue, X₄₁ is a V or I residue, X₄₂ is a K, T, or Q residue,X₄₃ is a L, V, or K residue, X₄₄ is a K, T, or E residue, X₄₅ is a K, P,or N residue, X₄₆ is a L, I, or M residue, X₄₇ is a K or N residue, X₄₈is a V or I residue, X₄₉ is a H or P residue, X₅₀ is a K or E residue,X₅₁ is a V or I residue, X₅₂ is a V or I residue, X₅₃ is a V or Presidue, X₅₄ is a T or S residue, X₅₅ is an E or D residue, X₅₆ is a Qor E residue, X₅₇ is an A or Q residue, X₅₈ is a S or G residue orrepresents a single residue deletion, X₅₉ is a K or N residue orrepresents a single residue deletion, X₆₀ is a H or Q residue, X₆₁ is aV or K residue, X₆₂ is a T or E residue, X₆₃ is a V or I residue, X₆₄ isa K or E residue, X₆₅ is an A or S residue, X₆₆ is a T or I residue, X₆₇is a V or T residue, X₆₈ is an I or E residue, X₆₉ is a T or P residue,X₇₀ is a G or E residue, X₇₁ is a L or V residue, X₇₂ is a T or Gresidue, X₇₃ is a L or M residue, X₇₄ is an R or E residue, X₇₅ is a Vor M residue, X₇₆ is a V or I residue, X₇₇ is a K or N residue, X₇₈ isan A, T, or S residue, X₇₉ is a L or M residue, X₈₀ is a K or R residue,X₈₁ is a L or F residue, X₈₂ is an A or S residue, X₈₃ is a T or Dresidue, X₈₄ is a S or E residue, X₈₅ is a T or E residue, X₈₆ is a V orP residue, X₈₇ is a T or H residue, X₈₈ is a H or R residue, X₈₉ is a Lor R residue, X₉₀ is a L or M residue, X₉₁ is a V or T N residue, X₉₂ isan A or P residue, X₉₃ is a K or R residue, X₉₄ is an E or D residue,X₉₅ is a V or T residue, X₉₆ is a T or S residue, X₉₇ is an A or Tresidue, X₉₈ is a V or I residue, X₉₉ is a Q or D residue, X₁₀₀ is a Sor M residue, X₁₀₁ is a S or G residue, X₁₀₂ is a S or D residue, X₁₀₃is a T or N residue, X₁₀₄ is a T residue or represents a single residuedeletion, X₁₀₅ is a L or T residue, X₁₀₆ is a L or I residue, X₁₀₇ is anA or T residue, X₁₀₈ is a K or R residue, X₁₀₉ is a L or V residue, X₁₁₀is a K or R residue, X₁₁₁ is a T or Q residue, X₁₁₂ is a V or M residue,X₁₁₃ is a V, T, or S residue, X₁₁₄ is a K or S residue, X₁₁₅ is a K or Qresidue, X₁₁₆ is a L or I residue, X₁₁₇ is a V or E residue, X₁₁₈ is a Vor I residue, X₁₁₉ is a V or I residue, X₁₂₀ is a L or V residue, X₁₂₁is a V or I residue, X₁₂₂ is a K, R, or Q residue, X₁₂₃ is a K, Q, or Eresidue, X₁₂₄ is a K or E residue, X₁₂₅ is a T, E, or D residue, X₁₂₆ isa G or D residue, X₁₂₇ is an A or S residue, X₁₂₈ is a L or F residue,X₁₂₉ is a S or E residue, X₁₃₀ is a T, I, or S residue, X₁₃₁ is a Q, E,or M residue, X₁₃₂ is a L, G, or E residue, X₁₃₃ is a K, Q, or Eresidue, X₁₃₄ is a K or G residue, X₁₃₅ is a V, K, or R residue, X₁₃₆ isa A, T, or H residue, X₁₃₇ is a V, H, or Q residue, X₁₃₈ is a L or Nresidue, X₁₃₉ is an A or V residue, X₁₄₀ is a V or I residue, X₁₄₁ is aT or I residue, X₁₄₂ is a K, E, or D residue, X₁₄₃ is an E or D residue,X₁₄₄ is a K, S, or E residue, X₁₄₅ is an A or L residue, X₁₄₆ is an E orD residue, X₁₄₇ is a Y or N residue, X₁₄₈ is a V or I residue, X₁₄₉ is aV or I residue, X₁₅₀ is a A, V, or I residue, X₁₅₁ is a G or E residue,X₁₅₂ is a E, P, or D residue, X₁₅₃ is a K, S, or G residue, X₁₅₄ is an Aor Q residue, X₁₅₅ is a K or T residue, X₁₅₆ is a L or I residue, X₁₅₇is a S, H, or N residue, X₁₅₈ is a F or Y residue, X₁₅₉ is a K or Rresidue, X₁₆₀ is a K or Q residue, X₁₆₁ is an A, T, or S residue, X₁₆₂is an A, Y, or M residue, X₁₆₃ is a K or R residue, X₁₆₄ is an E or Dresidue, X₁₆₅ is a L or V residue, X₁₆₆ is a L or V residue, X₁₆₇ is a Lor F residue, X₁₆₈ is a L or I residue, X₁₆₉ is an A or M residue, X₁₇₀is a L or V residue, X₁₇₃ is a V or I residue, X₁₇₄ is a T or G residue,X₁₇₅ is an A or T residue, X₁₇₆ is a M residue or represents a singleresidue deletion, X₁₇₇ is a F residue or represents a single residuedeletion, and X₁₇₈ is a G residue or represents a single residuedeletion. 51. SEQUENCE M X₁ X₂ X₃ F I L X₄ M L V X₅ P S X₆ X₇ M R C X₈ GX₉ X₁₀ N X₁₁ PATTERN 2 D F V E G X₁₂ S G X₁₃ X₁₄ W V D X₁₅ V L E H G X₁₆C V T T M A X₁₇ X₁₈ K P T L D X₁₉ E L X₂₀ K T X₂₁ X₂₂ X₂₃ X₂₄ X₂₅ A X₂₆L R X₂₇ X₂₈ C I E A X₂₉ X₃₀ X₃₁ N X₃₂ T T X₃₃ X₃₄ R C P T Q G E X₃₅ X₃₆X₃₇ X₃₈ E E Q D X₃₉ X₄₀ X₄₁ X₄₂ C X₄₃ X₄₄ X₄₅ X₄₆ V D R G W G N G C G LR F G K G X₄₇ X₄₈ X₄₉ T C A X₅₀ X₅₁ C X₅₂ X₅₃ X₅₄ X₅₅ E G X₅₆ X₅₇ V QX₅₈ E N L X₅₉ Y X₆₀ X₆₁ X₆₂ X₆₃ T X₆₄ H X₆₅ G X₆₆ X₆₇ H X₆₈ V G N X₆₉ TX₇₀ X₇₁ X₇₂ G X₇₃ X₇₄ X₇₅ X₇₆ I T P Q X₇₇ X₇₈ X₇₉ X₈₀ E X₈₁ X₈₂ L X₈₃X₈₄ Y G X₈₅ X₈₆ X₈₇ X₈₈ X₈₉ C S P R T G L D F N X₉₀ X₉₁ X₉₂ L X₉₃ X₉₄ MK X₉₅ K X₉₆ W X₉₇ V H X₉₈ Q W X₉₉ X₁₀₀ D L P L P W T X₁₀₁ G A X₁₀₂ TX₁₀₃ X₁₀₄ X₁₀₅ X₁₀₆ W N X₁₀₇ K E X₁₀₈ X₁₀₉ V T F K X₁₁₀ X₁₁₁ H A K X₁₁₂Q X₁₁₃ V X₁₁₄ V L G S Q E G X₁₁₅ M H X₁₁₆ A L X₁₁₇ G X₁₁₈ T E X₁₁₉ X₁₂₀X₁₂₁ X₁₂₂ X₁₂₃ G X₁₂₄ T X₁₂₅ X₁₂₆ F X₁₂₇ G X₁₂₈ L K C X₁₂₉ X₁₃₀ X₁₃₁ MX₁₃₂ K L X₁₃₃ X₁₃₄ K G X₁₃₅ S Y X₁₃₆ M C T G X₁₃₇ F X₁₃₈ X₁₃₉ X₁₄₀ K EX₁₄₁ AN E T Q H G T X₁₄₂ X₁₄₃ X₁₄₄ X₁₄₅ V X₁₄₆ Y X₁₄₇ G X₁₄₈ X₁₄₉ X₁₅₀ PC K I P X₁₅₁ X₁₅₂ X₁₅₃ X₁₅₄ D X₁₅₅ X₁₅₆ X₁₅₇ X₁₅₈ X₁₅₉ X₁₆₀ X₁₆₁ G R L IT X₁₆₂ N P X₁₆₃ V X₁₆₄ X₁₆₅ X₁₆₆ X₁₆₇ P V N I E X₁₆₈ E P P F G X₁₆₉ SX₁₇₀ I X₁₇₁ X₁₇₂ G X₁₇₃ X₁₇₄ X₁₇₅ X₁₇₆ X₁₇₇ L X₁₇₈ X₁₇₉ X₁₈₀ W X₁₈₁ X₁₈₂K G S S I G X₁₈₃ M F E X₁₈₄ T X₁₈₅ R G A X₁₈₆ R M AN I L G X₁₈₇ T A W DX₁₈₈ G S X₁₈₉ X₁₉₀ X₁₉₁ X₁₉₂ X₁₉₃ X₁₉₄ X₁₉₅ X₁₉₆ X₁₉₇ X₁₉₈ X₁₉₉ X₂₀₀X₂₀₁ X₂₀₂ X₂₀₃ X₂₀₄ X₂₀₅ X₂₀₆ X₂₀₇ X₂₀₈ X₂₀₉ X₂₁₀ X₂₁₁ X₂₁₂ X₂₁₃ whereinX₁ is an A, V, T, or G residue, X₂ is a V or I residue, X₃ is an I or Fresidue, X₄ is a L or M residue, X₅ is an A or T residue, X₆ is a Y or Mresidue, X₇ is an A, T, or G residue, X₈ is a V or I residue, X₉ is a V,I or T residue, X₁₀ is an S or G residue, X₁₁ is a R or G residue, X₁₂is a L or V residue, X₁₃ is a A or G residue, X₁₄ is an A, T, or Sresidue, X₁₅ is a L or V residue, X₁₆ is a S or G residue, X₁₇ is a K,R, or Q residue, X₁₈ is a G or N residue, X₁₉ is an I or F residue, X₂₀is a L, I, or or Q residue, X₂₁ is a T, I, or E residue, X₂₂ is a A or Vresidue, X₂₃ is a K or T residue, X₂₄ is a Q, E, or N residue, X₂₅ is aL, V, or P residue, X₂₆ is a L, V, or T residue, X₂₇ is a K or Tresidue, X₂₈ is a L or Y residue X₂₉ is an K or S residue, X₃₀ is an Lor I residue, X₃₁ is a T or S residue, X₃₂ is an I or T residue, X₃₃ isan A, E residue, or D residue, X₃₄ is a T or S residue, X₃₅ is an A or Presidue, X₃₆ is a I, T, Y, or N residue, X₃₇ is a L or M residue, X₃₈ isa K, V, or P residue, X₃₉ is a T or Q residue, X₄₀ is a Q or N residue,X₄₁ is a F or Y residue, X₄₂ is a V or I residue, X₄₃ is a K or Rresidue, X₄₄ is a H or R residue, X₄₅ is a T, S, or D residue, X₄₆ is aV, F, Y, or M residue, X₄₇ is a S or G residue, X₄₈ is a L, V, or Iresidue, X₄₉ is a V or I residue, X₅₀ is a K or M residue, X₅₁ is a K,T, or Q residue, X₅₂ is a L, V, or K residue, X₅₃ is a K, T, or Eresidue, X₅₄ is a K, P, or N residue, X₅₅ is a L I M residue, X₅₆ is a Kor N residue, X₅₇ is a V I residue, X₅₈ is a H, P, or Y residue, X₅₉ isa K or E residue, X₆₀ is a T S residue, X₆₁ is a V I residue, X₆₂ is a Vor I residue, X₆₃ is a V or I residue, X₆₄ is a V or P residue, X₆₅ is aT or S residue, X₆₆ is a E or D residue, X₆₇ is a Q or E residue, X₆₈ isan A or Q residue, X₆₉ is a E or D residue, X₇₀ is a T, S, or G residueor represents a single residue deletion, X₇₁ is a K E N or represents asingle residue deletion, X₇₂ is a H or Q residue, X₇₃ is a V, K, or Tresidue, X₇₄ is a T, I, or E residue, X₇₅ is a A, V or I residue, X₇₆ isa K, T, or E residue, X₇₇ is a A or S residue, X₇₈ is an S or P residue,X₇₉ is a T or I residue, X₈₀ is a V, T, or S residue, X₈₁ is an A or Iresidue, X₈₂ is an I, Q, or E residue, X₈₃ is a T or P residue, X₈₄ is aG, E, or D residue, X₈₅ is a A or T residue, X₈₆ is a L or V residue,X₈₇ is a T or G residue, X₈₈ is a L or M residue, X₈₉ is a E or Dresidue, X₉₀ is a R or E residue, X₉₁ is a V or M residue, X₉₂ is a V orI residue, X₉₃ is a L or M residue, X₉₄ is an K or T residue, X₉₅ is aK, S, or N residue, X₉₆ is a A, T, or S residue, X₉₇ is an L or Mresidue, X₉₈ is a K, R, or G residue, X₉₉ is a L or F residue, X₁₀₀ is aL or F residue, X₁₀₁ is an A or S residue, X₁₀₂ is a T, S, or D residue,X₁₀₃ is a S or E residue, X₁₀₄ is a V, T, Q, or E residue, X₁₀₅ is a V,H, E, or P residue, X₁₀₆ is a T or H residue or represents a singleresidue deletion, X₁₀₇ is a H or R residue, X₁₀₈ is a L or R residue,X₁₀₉ is a L or M residue, X₁₁₀ is a V, T, or N residue, X₁₁₁ is a A or Presidue, X₁₁₂ is a K or R residue, X₁₁₃ is a E or D residue, X₁₁₄ is a Vor T residue, X_(115 is an A T residue, X) ₁₁₆ is a T S residue, X₁₁₇ isan A or T residue, X₁₁₈ is an A or T residue, X₁₁₉ is a V or I residue,X₁₂₀ is a Q or D residue, X₁₂₁ is a T, S, N, M residue, X₁₂₂ is a S or Gresidue, X₁₂₃ is a S or D residue, X₁₂₄ is a T or N residue, X₁₂₅ is aL, T, or H residue, X₁₂₆ is a L, I, or M residue, X₁₂₇ is an A or Tresidue, X₁₂₈ is a H or R residue, X₁₂₉ is a K or R residue, X₁₃₀ is a Lor V residue, X₁₃₁ is a K or R residue, X₁₃₂ is an E or D residue, X₁₃₃is a T, R, or Q residue, X₁₃₄ is a L or I residue, X₁₃₅ is a V or Mresidue, X₁₃₆ is a V, T, or S residue, X₁₃₇ is a K or S residue, X₁₃₈ isa K or Q residue, X₁₃₉ is a L or I residue, X₁₄₀ is a V or E residue,X₁₄₁ is a V or I residue, X₁₄₂ is a V or I residue, X₁₄₃ is a L or Vresidue, X₁₄₄ is a V or I residue, X₁₄₅ is an K, R, or Q residue, X₁₄₆is a K, Q, or E residue, X₁₄₇ is a K or E residue, X₁₄₈ is a T, E, or Dresidue, X₁₄₉ is a G or D residue, X₁₅₀ is a A or S residue, X₁₅₁ is aL, V, or F residue, X₁₅₂ is a S or E residue, X₁₅₃ is a I, T, or Sresidue, X₁₅₄ is an Q, E, or M residue, X₁₅₅ is a L, G, or E residue,X₁₅₆ is a K, Q, or E residue, X₁₅₇ is a K or G residue, X₁₅₈ is a K, V,or R residue, X₁₅₉ is an A, T, or H residue, X₁₆₀ is a V, H, or Qresidue, X₁₆₁ is a L or N residue, X₁₆₂ is an A or V residue, X₁₆₃ is aA, V, or I residue, X₁₆₄ is an I or T residue, X₁₆₅ is a K, E, or Dresidue, X₁₆₆ is a E or D residue, X₁₆₇ is a K, S, or E residue, X₁₆₈ isa L or A residue, X₁₆₉ is an E or D residue, X₁₇₀ is a Y or N residue,X₁₇₁ is a V or I residue, X₁₇₂ is a V or I residue, X₁₇₃ is an A, V, orI residue, X₁₇₄ is a G or E residue, X₁₇₅ is an E, P, or D residue, X₁₇₆is a K, S, or G residue, X₁₇₇ is a A or Q residue, X₁₇₈ is a K or Tresidue, X₁₇₉ is a L or I residue, X₁₈₀ is a S, H, or N residue, X₁₈₁ isa F or Y residue, X₁₈₂ is a K or R residue, X₁₈₃ is a K or Q residue,X₁₈₄ is an A, T, or S residue, X₁₈₅ is an A, Y, or M rsidue, X₁₈₆ is a Kor R residue, X₁₈₇ is an E or D residue, X₁₈₈ is a L or F residue, X₁₈₉is an A, L, V, or I residue, X₁₉₀ is a G or Y residue, X₁₉₁ is a T or Gresidue, X₁₉₂ is a L or V residue or represents a single residuedeletion, X₁₉₃ is a L or F residue or represents a single residuedeletion, X₁₉₄ is a T or N residue or represents a single residuedeletion, X₁₉₅ is a S residue or represents a single residue deletion,X₁₉₆ is a L, V, or I residue or represents a single residue deletion,X₁₉₇ is a G residue or represents a single residue deletion, X₁₉₈ is a Kresidue or represents a single residue deletion, X₁₉₉ is an A or Mresidue or represents a single residue deletion, X₂₀₀ is a L or Vresidue or represents a single residue deletion, X₂₀₁ is a H residue orrepresents a single residue deletion, X₂₀₂ is a Q residue or representsa single residue deletion, X₂₀₃ is a V or I residue or represents asingle residue deletion, X₂₀₄ is a F residue or represents a singleresidue deletion, X₂₀₅ is a G residue or represents a single residuedeletion, X₂₀₆ is an A or S residue or represents a single residuedeletion, X₂₀₇ is a V or I residue or represents a single residuedeletion, X₂₀₈ is a F or Y residue or represents a single residuedeletion, X₂₀₉ is a T or G residue or represents a single residuedeletion, X₂₁₀ is an A, T, or S residue or represents a single residuedeletion, X₂₁₁ is a V or M residue or represents a single residuedeletion, X₂₁₂ is a G or F residue or represents a single residuedeletion, and X₂₁₃ is a K or G residue or represents a single residuedeletion. 52. DEN-1 MGIIFILLMLVTPSMA PRM15 53. DEN-2 MGLILILQTAVAPSMTPRM15 54. DEN-3 MVVIFILLMLVTPSMT PRM15 55. DEN-4 MTVFFILMMLVAPSYG PRM1556. 6C6 PRM15 MTVFFILMMLVTPSMA 57. PRM15 6F4 MAVFFILLMLVTPSMT 58. PRM157A9 MTVFFILMMLVAPSYA 59. PRM15 MTVFFILLMLVAPSYG 11E2 60. PRM15MAVFFILMMLVAPSYG 12E3 61. PRM15 MGIIFILLMLVTPSYG 15C2 62. PRM15MTVFFILMMLVTPSMT 17A12 63. PRM15 MTVFFILLMLVTPSMT 17D5 64. PRM15MTVFFILMMLVAPSMA 17G5 65. 2/7MVVIFILLMLVTPSMTMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELIKTEATQPATLRKYCIEAKLTNTTTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 66. 2G11MTVFFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 67. 5/21MTVFFILMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGESYIVVGVGDSALTLHWFRKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGA 68. 6C6MTVFFILMMLVTPSMAMRCVGIGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCLEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMILMKMKNKAWMVHRQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGMFEATARGARRMAILGDTAWDFGSAYT 69. 6E12MTVFFILMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGTTTIFAGHLKCKVRMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFG 70. 6F4MAVFFILLMLVTPSMTMRCVGIGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDFELIKTEATQPATLRKYCIEAKLTNTTTESRCPTQGEAILPEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFEATARGARRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 71. 7A9MTVFFILMMLVAPSYAMRCVGIGNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEASISNITTATRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMILLTMKSKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKNAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 72. 11B1MVVIFILLMLVTPSMTMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELIKTEATQPATLRKYCIEAKLTNTTTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHTALTGATEVDSGDGNHMFAGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITANPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFRKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGAIYGA 73. 11B8MTVFFILLMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKLTNTTTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNHMFAGHLKCKVRMEKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGQMFEATARGARRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFG 74. 11C4MTVFFILMMLVAPSYGMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRTYCIEASISNITTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMILLTMKSKTWLVHKQWFLDLPLPWTAGADTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVGDKALKLHWFKKGSSIGKMFEATARGAKRMAILGETAWDFGSAYT 75. 11C11MTVFFILMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEASISNITTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKTWLVHKQWFLDLPLPWTAGADTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGEDAPCKIPFSTEDGQGKAHNGRLITANPIVTEKDSPVNIEAEPPFGDSYIVVGAGEKALKLHWFKKGSSIGQMFEATARGARRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 76. 11E2MTVFFILLMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKRQDVTVLGSQEGAMHSALTGATEIQMSSGNLLFAGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGESYIVVGVGDSALTLHWFRKGSSIGQMFEATARGAKRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 77. 12E3MAVFFILMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGESYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMF G 78. 12H4MTVFFILMMLVAPSYAMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELIKTEATQPATLRKYCIEASISNITTDSRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFEATARGARRMAILGDTAWDFGSAYT 79. 13E2MTVFFILMMLVTPSMAMRCVGIGNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGTDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGKMFEATARGAKRMAILGETAWDFGSVGGLLTSLGKMVHQIFGSVYTTMF G 80. 13E11MTVFFILMMLVTPSMAMRCVGVGNRDFVEGVSGGAWVDVVLEHGGCVTTMAKNKPTLDIELIKTEATQPATLRKYCIEASISNITTDTRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGTVTMECSPRTGLDFNEMILLTMKSKTWLVHKQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGTDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGESYIVVGAGEKALTLHWFRKGSSIGQMFETTMRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 81. 13F11MTVFFILMMLVTPSMAMRCVGIGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDIELIKTEATQPATLRKYCIEAKITNITTDSRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDEKGKAHNGRLITANPIVIDKEKPVNIELEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFESTYRGAKRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 82. 14B1MTVFFILMMLVAPSYGMRCVGISNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTDSRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGNIVQPENLKYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGADTETPTWNRKELLVTFKNAHAKRQDVTVLGSQEGAMHSALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFESTARGARRMAILGDTAWDFGSAYT 83. 14E9MTVFFILLMLVTPSMAMRCVGIGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDIELIKTEATQPATLRKYCIEAKITNITTDSRCPTQGEAILPEEQDQQYICRRDVVDRGWGNGCGLFGKGSVVTCAKFQCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILMKMKNKAWMVHKQWFLDLPLPWTSGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSAYT 84. 14G10MAVFFILLMLVTPSMAMRCIGISNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTDTRCPTQGEAILPEEQDQQYICRRDVVDRGWGNGCGLFGKGSLVTCAKFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 85. 14H2MTVFFILMMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEAKITNITTATRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFQCVTKLEGNIVQPENLEYTIVITPHSGEEHAVGNDTQGVTVEITPQASTVEAILTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYKGEDAPCKIPFSSQDGQGKAHNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFETTMRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 86. 15C2MVVIFILLMLVTPSMAMRCVGISNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTDSRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKIVQYENLKYSVIVTVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCRLKMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGESYIVIGVEPGQLKLHWFKKGSSIGQMFEATARGARRRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 87. 15D4MRVFFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCLEPIEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGA 88. 15H4MTVFFILMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFELIKTEVTNPATLRKYCIEASISNITTATRCPTQGEANLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGRLKCRLKMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFESTYRGAKRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSVYTTMFG 89. 16B4MGIIFILLMLVTPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEASISNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGTTTIFAGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGTTTIFAGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGVGDKALKINWYKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFG 90. 16E8MTVFFILMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWFLDLPLPWTSGATTETPTWNRKELLVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFETTMRGAKRMAILGDTAWDFGSVGGVFTSIGKALHQVFGAIYGA 91. 16E10MTVFFILMMLVAPSYGMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKISNTTTDSRCPTQGEATLVEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGALGLECSPRTGLDFNEMILLTMKNKAWMVHGQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGESYIVVGVGDSALTLHWFRKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSVYTTMFG 92. 16F12MTVFFILMMLVAPSYGMRCVGVGNGDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFELIKTIAKEVALLRTYCIEASISNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGTMHTALTGATEIQMSSGTTTIFAGHLKCKVKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGESYIVVGVGDSALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 93. 16G11MGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEASISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSLGGLLTSLGKAVHQVFGSVYTTMFG 94. 17A12MTVFFILMMLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFLEIKTEATQPATLRKYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCLEPIEGKVVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTSGATTETPTWNRKELLVTFKNAHAKRQDVTVLGSQEGAMHSALTGATEIQTSGTTTIFAGHLKCRVRMDKLQLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPVSSQDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAWDLGSVGGVFTSIGKALHQVFGAIYGA 95. 17D5MTVFFILLMLVTPSMTMRCIGISNRDFVEGVSGATWVDVVLEHGGCVTTMARNKPTLDIELIKTEATQPATLRKYCIEASISNITTATRCPTQGEAILPEEQDQQYICRRDVVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTSGATTETPTWNRKELLVTFKNQHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGA 96. 17D11MTVFFILMMLVAPSYGMRCVGTGNRDFVEGLSGATWVDVVLEHGGCVTTMAQGKPTLDIELIKTEATQPATLRKYCIEAKLTNTTTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 97. 17F5MTVFFILMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPALTRKYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGVGDSALTLHWFRKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSLGKAVHQIFGSVYTTMFG 98. 17F11MVVIFILLMLVTPSMTMRCIGISNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWLLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 99. 17G5MTVFFILMMLVAPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEAILKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQEVVVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMDKLTLKGVSYVMCTGSFKLEKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 100. 17H3MVVIFILLMLVTPSMTMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKTWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKRQDVTVLGSQEGAMHSALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPAVTDKEKPVNIELEPPFGDSYIVVGAGDKALKLSWFKKGSSIGQMFESTYRGAKRMAILGETAWDLGSIGGVFTSVGK 101. 17H10MVVIFILLMLVTPSMTMRCIGISNRDFVEGVSGGSWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTDSRCPTQGEAIMPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFTCKKNMEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGVLTSLGKMVHQIFGSVYTTMFG 102. 17H12MVVIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAQGKPTLDIELLKTEVTNPATLRKYCIEAKLTNTTTESRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPIVIDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMF G 103. 18A9MTVFFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTATRCPTQGEAILPEEQDQNYVCKHSMVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLTLKGVSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALTLHWFRKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVFTSVGK 104. 18B7MTVFFILLMLVTPSMAMRCIGISNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 105. 18D7MVVIFILLMLVTPSMTMRCIGISNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGTTTIFAGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTILIKVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 106. 18E9MTVFFILMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTEATQPATLRKYCIEAKLTNTTTATRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYSVIVTVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRKELLVTFKNAHAKRQDVTVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSVYTTMFG 107. 18E10MTVFFILLMLVTPSMAMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKYCIEAKLTNTTTESRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDSCPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKAVHQVFGSVYTTMF G 108. 18E11MTVFFILMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTVKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGKAHNGRLITANPIVIDKEKPVNIEAEPPFGESYIVVGAGDKALKLSWFKKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGVFTSIGKALHQVFGAIYGA 109. 18H2MTVFFILMMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTSNHGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKKQDVVVLGSQEGAMHTALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMF G 110. 18H6MVVIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFTCKKNMEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLTLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKNAHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTEDGQGKAHNGRLITVNPIVIDKEKPVNIELEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGAIYGA 111. 21C1MALIFILLTAVAPSMTMRCIGISNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLD PRM15FELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRG ETRUNCWGNGCGLFGKGGVVTCAKFKCVTKLEGKIVQYENLEYTVVVTVHNGDTHAVGNDTSNH(“E-truncated”GVTATITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDL or “tE”)_(—)PLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMDKLQLKGMSYSMCTGKFQLVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGDSYIIIGVGDSALTLHWFRKGSSIGKMFESTYRGARRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMF G 112. 23C12MALIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLD PRM15IELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRG ETRUNCWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFRKGSSIGQMFETTMRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 113. 23D5MGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLD PRM15IELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVESQDTNFVCRRTFVDRG ETRUNCWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHNGDTHAVGNDTQGVTVEITPQAPTSEIQLTDYGALTLDSCPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGTTEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTARGARRMAILGDTAWDFGSIGGVFTSVGK 114. 23F5MTVFFVLMMLVAPSMAMRCVGIGNRDFVEGLSGGAWVDLVLEHGGCVTTMAKNKPTLD PRM15FELIKTEATQPATLRKYCIEAKLTNTTTESRCPTQGEAILPEEQDQNYVCKHTYVDRG ETRUNCWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKTWLVHKQWFLDLPLPWTAGADTREVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCRLKMDKLELKGMSYSMCTGKFRIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSTGGVFTSVGK 115. 23G3MGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLD PRM15IELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRG ETRUNCWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGESNIVIGIGDKALKINWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSIGKALHQVFGAIYGA 116. 23H7MGIIFILLMLVTPSMTMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLD PRM15IELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRG ETRUNCWGNGCGLFGKGSLITCAKFKCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFTGHLKCRLKMDKLTLKGVSYVTCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGK 117. DEN-1MRSVTMILMLLPTALAFHLTTRGGEPTLIVSKQERGKSLLFKTSAGVNMCTLIAMDLG REST OFELCEDTMTYKCPRMTEAEPDDVDCWCNATDTWVTYGTCSQTGEHRRDKRSVALDPHVG C15/PRMLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQK 118. DEN-2MNRRRRTVGVIIMLIPTAMAGHLTTRNGEPHMIVGRQEKGKSLLFKTEDGVNMCTLMA REST OFIDLGELCEDTITYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCTTTGEHRREKRSVALV C15/PRMPHVGMGLETRTETWMSSEGAWKHAQRIETWILRHPGFIIMAAILAYTIGTTHFQR 119. DEN-3MKTSLCLMMMLPATLAFHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLG REST OFEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVG C15/PRMMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSLTQK 120. DEN-4MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG REST OFEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSG C15/PRMMGLETRAETWMSSEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQR 121. 21C1 RESTMNRRRRTVGVIIMLIPTAMAFHLTTRNGEPHMIVGRQEKGKSLLFKTEDGVNMCTLMA OFIDLGELCEDTVTYKCPLLVNTEPEDIDCWCNLTSAWVTYGTCNQAGEHRRDKRSVALA C15/PRMPHVGMGLETRTETWMSSEGAWKHAQRIETWILRHPGFIIMAAILAYTIGTTHFQR 122. 23C12MRSVTMILMLLPTALAFHLTTRGGEPTLIVSKQERGKSLLFKTASGINMCTLIAMDLG REST OFEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALVPHVG C15/PRMMGLETRTETWMSSEGAWKHAQRIETWILRHPGFIIMAAILAYTIGTTHFQR 123. 23D5 RESTMKTSLCLMMMLPATLAFHLTSRDGEPRMIVGKNERGKSLLFKTEDGVNMCTLIAMDLG OFELCEDTMTYKCPRMTEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVG C15/PRMMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSITQK 124. 23F5 RESTMRSAITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG OFEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSG C15/PRMMGLDTRTQTWMSAEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQR 125. 23G3 RESTMKTSLCLMMMLPATLAFHLTTRNGEPHMIVGRQEKGKSLLFKTSAGVNMCTLIAMDLG OFELCEDTMTYKCPRMTEAEPEDIDCWCNLTSTWVTYGTCTQSGERRREKRSVALTPHSG C15/PRMMGLETRAETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQK 126. 23H7 RESTMKTSLCLMMMLPATLAFHLTSRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG OFEMCEDTVTYKCPLLRQNEPDDVDCWCNATDTWVTYGTCSQTGEHRRDKRSVALDPHVG C15/PRMLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQK 127. DEN-1LIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVM REST OF VQAENV 128. DEN-2 AFSGVSWTMKILIGVIITWIGMNSRSTSLSVSLVLVGVITLYLGAMVQA REST OFENV 129. DEN-3 ALFSGVSWIMKIGIGVLLTWIGLNSKNTSMSFSCIAIGIITLYLGVVVQA RESTOF ENV 130. DEN-4 GVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA REST OFENV 131. 21C1 REST GVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGAMVQA OF ENV132. 23C12 GVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA REST OF ENV133. 23D5 restLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVV of ENV VQA134. 23F5 restLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVM of ENV VQA135. 23G3 rest AFSGVSWTMKILIGVIITWIGMNSRSTSLSVSLVLVGVITLYLGAMVQA of ENV136. 23H7 RESTLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGAM OF ENV VQA137. SEQ G V S W X₁ X₂ X₃ I X₄ I G X₅ X₆ X₇ X₈ W X₉ G X₁₀ N S X₁₁ X₁₂ TPATTERN S X₁₃ X₁₄ X₁₅ X₁₆ X₁₇ X₁₈ X₁₉ X₂₀ G X₂₁ X₂₂ T L X₂₃ L G X₂₄ X₂₅V X₂₆ REST OF A, ENV whereine X₁ is a M, I, or T residue, X₂ is a V or Mresidue, X₃ is a K or R residue, X₄ is a L or G residue, X₅ is a V, I,or F residue, X₆ is a I or L residue, X₇ is an I, L, or V residue, X₈ isa T or L residue, X₉ is an I or L residue, X₁₀ is a M, L, or T residue,X₁₁ is a K or R residue, X₁₂ is a S or N residue, X₁₃ is a L or Mresidue, X₁₄ is a S or A residue, X₁₅ is a V, M, or F residue, X₁₆ is aS or T residue, X₁₇ is a L or C residue, X₁₈ is a V or I residue, X₁₉ isa L or A residue, X₂₀ is a V or I residue, X₂₁ is a V, M, I, or Gresidue, X₂₂ is a V or I residue, X₂₃ is a Y or F residue, X₂₄ is an A,V, or F residue, X₂₅ is a M, V, or T residue, and X₂₆ is a Q or Hresidue. 138. SEQ X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ P X₁₂ X₁₃ X₁₄ A FX₁₅ L X₁₆ X₁₇ PATTERN R X₁₈ G E P X₁₉ X₂₀ I V X₂₁ X₂₂ X₂₃ E X₂₄ G X₂₅X₂₆ L L F K T X₂₇ REST OF X₂₈ G X₂₉ N X₃₀ C T L X₃₁ A X₃₂ D L G E X₃₃ CX₃₄ D T X₃₅ T Y K C15/PRM C P X₃₆ X₃₇ X₃₈ X₃₉ X₄₀ E P X₄₁ D X₄₂ D C W CN X₄₃ T X₄₄ X₄₅ W V X₄₆ Y G T C X₄₇ X₄₈ X₄₉ G E X₅₀ R R X₅₁ K R S V A LX₅₂ P H X₅₃ G X₅₄ G L X₅₅ T R X₅₆ X₅₇ T W M S X₅₈ E G A W X₅₉ X₆₀ X₆₁X₆₂ X₆₃ X₆₄ E X₆₅ W X₆₆ L R X₆₇ P X₆₈ F X₆₉ X₇₀ X₇₁ A X₇₂ X₇₃ X₇₄ A X₇₅X₇₆ I G X₇₇ X₇₈ X₇₉ X₈₀ X₈₁, wherein X₁ is a M or R residue, X₂ is a Ror K residue, X₃ is a S or T residue, X₄ is a V, S, T, or A residue, X₅is a T, L, G, or I residue, X₆ is a M, C, V, or T residue, X₇ is an I orL residue, X₈ is a L, M, or I residue, X₉ is a M or C residue, X₁₀ is aL or M residue, X₁₁ is a L or I residue, X₁₂ is a T or A residue, X₁₃ isan A, T, or V residue, X₁₄ is a L or M residue, X₁₅ is a H or S residue,X₁₆ is a T or S residue, X₁₇ is a T or S residue, X₁₈ is a G, D, or Nresidue, X₁₉ is a T, L, R, or H residue, X₂₀ is a L or M residue, X₂₁ isa S, A, or G residue, X₂₂ is a K or R residue, X₂₃ is a Q, H, or Nresidue, X₂₄ is a R or K residue, X₂₅ is a K or R residue, X₂₆ is a S orP residue, X₂₇ is a S, T, A, or E residue, X₂₈ is an A, E, S, or Dresidue, X₂₉ is a V or I residue, X₃₀ is a M or K residue, X₃₁ is an Ior M residue, X₃₂ is a M or I residue, X₃₃ is a L or M residue, X₃₄ isan E or D residue, X₃₅ is a M, V, or I residue, X₃₆ is a R, L, or Hresidue, X₃₇ is a M, L, or I residue, X₃₈ is a T, R, or V residue, X₃₉is an E, Q, or N residue, X₄₀ is an A, N, V, or T residue, X₄₁ is a D orE residue, X₄₂ is a V or I residue, X₄₃ is an A, L, or S residue, X₄₄ isa D or S residue, X₄₅ is a T or A residue, X₄₆ is a T or M residue, X₄₇is a S, N, or T residue X₄₈ is a Q or T residue, X₄₉ is a T, A, or Sresidue, X₅₀ is a H or R residue, X₅₁ is a D or E residue, X₅₂ is a D,A, T, or V residue, X₅₃ is a V or S residue, X₅₄ is a L or M residue,X₅₅ is an E or D residue, X₅₆ is a T or A residue, X₅₇ is an E or Qresidue, X₅₈ is a S or A residue, X₅₉ is a K or R residue, X₆₀ is a H orQ residue, X₆₁ is an I, V, or A residue, X₆₂ is a Q or E residue, X₆₃ isa K or R residue, X₆₄ is a V or I residue, X₆₅ is a T or S residue, X₆₆is an A or I residue, X₆₇ is a H or N residue, X₆₈ is a G or R residue,X₆₉ is a T, I, or A residue, X₇₀ is a V, I, or L residue, X₇₁ is an I,L, or M residue, X₇₂ is a L, A, or G residue, X₇₃ is a F or I residue,X₇₄ is a L or M residue, X₇₅ is a H or Y residue, X₇₆ is an A, Y, T, orM residue, X₇₇ is a T or Q residue, X₇₈ is a T or S residue, X₇₉ is anI, L, H, or G residue, X₈₀ is a T, F, or I residue, and X₈₁ is a K or Rresidue. 139. 2G11-D4MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLKMDKLQLKGMSYTMCTGKFQIVKEIAETQHGTILIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA 140. 5/21-D1MRSVTMILMLLPTALAFHLTTRGGEPTLIVSKQERGKSLLFKTSAGVNMCTLIAMDLGELCEDTMTYKCPRMTEAEPDDVDCWCNATDTWVTYGTCSQTGEHRRDKRSVALDPHVGLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGESYIVVGVGDSALTLHWFRKGSSIGQMFETTMRGAKRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQA 141. 6E12-D4MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEKHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGTTTIFAGHLKCKVRMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSVGGLLTSLGKMVHQIFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA 142. 21C1MNRRRRTVGVIIMLIPTAMAFHLTTRNGEPHMIVGRQEKGKSLLFKTEDGVNMCTLMA (C15/fullIDLGELCEDTVTYKCPLLVNTEPEDIDCWCNLTSAWVTYGTCNQAGEHRRDKRSVALA prM/fullPHVGMGLETRTETWMSSEGAWKHAQRIETWILRHPGFIIMAAILAYTIGTTHFQRALI E)FILLTAVAPSMTMRCIGISNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFKCVTKLEGKIVQYENLEYTVVVTVHNGDTHAVGNDTSNHGVTATITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMDKLQLKGMSYSMCTGKFQLVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGDSYIIIGVGDSALTLHWFRKGSSIGKMFESTYRGARRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGAMVQA 143. 23C12MRSVTMILMLLPTALAFHLTTRGGEPTLIVSKQERGKSLLFKTASGINMCTLIAMDLG (C15/fullEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALVPHVG prM/fullMGLETRTETWMSSEGAWKHAQRIETWILRHPGFIIMAAILAYTIGTTHFQRALIFILL E)MLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFRKGSSIGQMFETTMRGAKRMAIIGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA 144. 23D5MKTSLCLMMMLPATLAFHLTSRDGEPRMIVGKNERGKSLLFKTEDGVNMCTLIAMDLG (C15/fullELCEDTMTYKCPRMTEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVG prM/fullMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSITQKGIIFILL E)MLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHNGDTHAVGNDTQGVTVEITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGTTEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVVVQA 145. 23F5MRSAITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG (C15/fullEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSG prM/fullMGLDTRTQTWMSAEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQRTVFFVLM E)MLVAPSMAMRCVGIGNRDFVEGLSGGAWVDLVLEHGGCVTTMAKNKPTLDFELIKTEATQPATLRKYCIEAKLTNTTTESRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKTWLVHKQWFLDLPLPWTAGADTREVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCRLKMDKLELKGMSYSMCTGKFRIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQA 146. 23G3MKTSLCLMMMLPATLAFHLTTRNGEPHMIVGRQEKGKSLLFKTSAGVNMCTLIAMDLG (C15/fullELCEDTMTYKCPRMTEAEPEDIDCWCNLTSTWVTYGTCTQSGERRREKRSVALTPHSG prM/fullMGLETRAETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGESNIVIGIGDKALKINWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVSLVLVGVITLYLGAMVQA 147. 2/7-D1MRSVTMILMLLPTALAFHLTTRGGEPTLIVSKQERGKSLLFKTSAGVNMCTLIAMDLG extendedELCEDTMTYKCPRMTEAEPDDVDCWCNATDTWVTYGTCSQTGEHRRDKRSVALDPHVG with D1LGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILL toMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELIKTEA C15/fullTQPATLRKYCIEAKLTNTTTESRCPTQGEPYLKEEQDQNYVCKHTYVDRGWGNGCGLF prM/fullGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITP E withQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGA DEN-1STSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGH aminoLKCRLKMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTE acidDGQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSI residuesGKMFESTYRGAKRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQA 148. 23H7MKTSLCLMMMLPATLAFHLTSRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG (C15/fullEMCEDTVTYKCPLLRQNEPDDVDCWCNATDTWVTYGTCSQTGEHRRDKRSVALDPHVG prM/fullLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILL E)MLVTPSMTMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFTGHLKCRLKMDKLTLKGVSYVTCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGAMVQA 149. DEN-1MGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLD PRM15/IELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRG trunc E-WGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEH (truncatedGTIATITPQAPTSEIQLTDYGALTLDSCPRTGLDFNRVVLLTMKKKSWLVHKQWFLDL envelope)PLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSG parentTTTIFAGHLKCRLKMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDLGSIGGVFTSVGK 150. DEN-2MGLILILQTAVAPSMTMRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLD PRM15/FLEIKTEATQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRG trunc EWGNGCGLFGKGGIVTCAMFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKH parentGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEIVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNRIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGA 151. DEN-3MVVIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLD PRM15/IELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRG trunc EWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGV ParentTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYT 152. DEN-4MTVFFILMMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLD PRM15/FELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRG trunc EWGNGCGLFGKGGVVTCAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNH ParentGVTATITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMF G 153. 23D5MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRK ETRUNCLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITC (“truncAKFKCVTKLEGKIVQYENLKYSVIVTVHNGDTHAVGNDTQGVTVEITPQAPTSEIQLT E” orDYGALTLDCSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQ “truncateDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGTTEIQTSGTTTIFAGHLKCRLKMDKL d E”)TLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTGAPCKVPIEIRDVNKEKVVGR only (noIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTARG prM)ARRMAILGDTAWDFGSIGGVFTSVGK 154. 23G3MRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRK ETRUNCLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC (“truncAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILP E”) onlyEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRK (no prM)ELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGESNIVIGIGDKALKINWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSIGKALHQVFGAIYGA 155. 23H7MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRK ETRUNCLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITC only (noAKFKCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILP prM)EYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFTGHLKCRLKMDKLTLKGVSYVTCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGK 156. 2/7atggtggtgatcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgca Round Itcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 157. 2G11atgaccgtgttcttcatcctgctgatgctggtgaccccctctatggccatgaggtgcg Round Itgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaacccatcgtgaccgagaaggacagccccgtgaacatcgaaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgttcggc 158 5/21atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round Itgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 159. 6E12atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round Itgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaccaccaccatcttcgccggccacctgaagtgcaaggtgaggatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagcccccctttggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttcggc 160. 6C6atgaccgtgttcttcatcctgatgatgctggtgaccccctctatggccatgaggtgcg Round Itgggcatcggcaaccgcgacttcgtggagggcgtgagcggcggcgcctgggtggacct shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcggcatcgtgacctgcgccatgttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgatgaagatgaagaacaaggcctggatggtgcacaggcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagtcactgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgcctacacc 161. 6F4atggccgtgttcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgcg Round Itgggcatcggcaaccgcgacttcgtggagggcgtgagcggcggcgcctgggtggacct shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgcttgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggagaagctgcgcatcaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 162. 7A9atgaccgtgttcttcatcctgatgatgctggtggccccctcctacgccatgaggtgcg Round Itgggcatcggcaaccgcgacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgcagaagaccgaggccacccagctggccaccctgaggaagctgtgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccaccctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagagcaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcaccaccgagatccagaacagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgttcggc 163. 11E2atgaccgtgttcttcatcctgctgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctactgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 164. 12E3atggccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggccgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcaccctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgttc ggc 165. 13E2atgaccgtgttcttcatcctgatgatgctggtgaccccctctatggccatgaggtgcg Round IItgggcatcggcaaccgcgacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttc ggc 166. 14E9atgaccgtgttcttcatcctgctgatgctggtgaccccctctatggccatgaggtgcg Round IItgggcatcggcaaccgcgacttcgtggagggcgtgagcggcggcgcctgggtggacct shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgatgaagatgaagaacaaggcctggatggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgcctacacc 167. 13E11atgaccgtgttcttcatcctgatgatgctggtgaccccctctatggccatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcgtgagcggcggcgcctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccagcatcagcaacatcaccaccgacaccaggtgccccacccagggtgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagagcaagacctggctagtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 168. 16E8atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 169. 16E10atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaaaatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccaccctggtggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcgccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacgggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaacagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttcggc 170. 18E9atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgcttgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaacagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttcggc 171. 18E10atgaccgtgttcttcatcctgctgatgctggtgaccccctctatggccatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgcagaagaccgaggccacccagctggccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgttc ggc 172.18E11 atgaccgtgttcttcatcctgctgatgctggtgaccccctctatggccatgaggtgcg RoundII tgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgcagaagaccgaggccacccagctggccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgaggccatcctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgcttgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 173. 11B1atggtggtgatcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgatacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgtagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaggaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcgccatctacggcgcc 174. 11B8atgaccgtgttcttcatcctgctgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggagaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttcggc 175. 11C4atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgagagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagagcaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgactgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtgggcgacaaggccctgaagctgcactggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgcctacacc 176. 11C11atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacatcaccaccgagagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgcttgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcgtggtgggcgccggcgagaaggccctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 177. 12H4atgaccgtgttcttcatcctgatgatgctggtggccccctcctacgccatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccagcatcagcaacatcaccaccgacagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggtgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgcctacacctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtgggcgacaaggccctgaagctgcactggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgcctacacc 176. 11C11atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccacctgggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacatcaccaccgagagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgaggacgccccctgcaagcggcgacagctacatcgtggtgggcgccggcgagaaggccctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 177. 12H4atgaccgtgttcttcatcctgatgatgctggtggccccctcctacgccatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccagcatcagcaacatcaccaccgacagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgcttgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggtgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgcctacacc 178. 13F11atgaccgtgttcttcatcctgatgatgctggtgaccccctctatggccatgaggtgcg Round IItgggcatcggcaaccgcgacttcgtggagggcgtgagcggcggcgcctgggtggacct shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacgagaagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 179. 14B1atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcatcagcaacagggacttcgtggagggcgtgagcggcggcgcctgggtggacct shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctgaagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgcttgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgcctacacc 180. 14G10atggccgtgttcttcatcctgctgatgctggtgaccccctctatggcaatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 181. 14H2atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcgtgagcggcggcgcctgggtggacct shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccaagatcaccaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgcttgaccttcaaggtgccccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacaagggcgaggacgccccctgcaagcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 182. 15C2atggtggtgatcttcatcctgctgatgctggtgaccccctctatggccatgaggtgcg Round IItcggcatcagcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgttcggc 183. 15D4atgaccgtgttcttcatcctgctgatgctggtgaccccctctatggccatgaggtgcg Round IItgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcctggagcccatcgagggcaaggtggtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgacgagaaggagcagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaagtgttcggcgccatctacggcgcc 184. 15H4atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccaacctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccgcctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagcccccctttggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaacagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttcggc 185. 16B4atgggcatcatcttcatcctgctgatgctggtgaccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgcttgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaccaccaccatcttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttcggc 186. 16F12atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacggggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccatcgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcaccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaccaccaccatcttcgccggccacctgaagtgcaaggtgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 187. 16G11atgggcatcatcttcatcctgctgatgctggtgaccccctctatggccatgaggtgcg Round IItgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccaccctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgcttgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcctgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgttcggc 188. 17A12atgaccgtgttcttcatcctgatgatgctggtgaccccctccatgacaatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcctggagcccatcgagggcaaggtggtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgcttgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctgaccggcgccaccgagatccagaccagcggcaccaccaccatcttcgccggccacctgaagtgcagggtgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacctcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 189. 17D5atgaccgtgttcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaggaacaagcccaccctggac cloneatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 190. 17D11atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcaccggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggcccagggcaagcccaccctggac cloneatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 191. 17F5atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacgacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcctgggcaaggccgtgcaccagatcttcggcagcgtgtacaccaccatgttcggc 192. 17F11atggtggtgatcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggctcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctaggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgttcggc 193. 17G5atgaccgtgttcttcatcctgatgatgctggtggccccctctatggccatgaggtgcg Round IItgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 194. 17H3atggtggtgatcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaagaagacctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccgcggtgaccgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacctgggcagcatcggcggcgtgttcaccag cgtgggcaag195. 17H10 atggtggtgatcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgcaRound II tcggcatcagcaacagggacttcgtggagggcgtgagcggcggcagctgggtggacgtshuffled ggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacclone atcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcatgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcacctgcaagaagaacatggagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttcggc 196. 17H12atggtggtgatcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggcccagggcaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttc ggc 197. 18A9atgaccgtgttcttcatcctgctgatgctggtgaccccctctatggccatgaggtgcg Round IItgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacagcatggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgcttgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcatccggccacctgaagtgcaggctgaggatggacaagctgacctgaagggcgtgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgttcaccagcgtgggcaag 198. 18B7atgaccgtgttcttcatcctgctgatgctggtgaccccctctatggcaatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac clonettcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgttcggc 199. 18D7atggtggtgatcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgca Round IItcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgt shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatgggaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaccaccaccatcttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgttcggc 200. 18H2atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg Round IItgggcgtgggcaacagggacttcgtggagggcgtgagcggcggcgcctgggtggacct shuffledggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggac cloneatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacaccagcaaccacggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccgtggtgaccaagaaggaggagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccatgttc ggc 201.5/21-D1 atgagatctgtgaccatgattctcatgctgctgcctactgctctggccttccatctga RoundI caacaagaggtggcgagcctaccctgatcgtgtccaagcaagaacgcggcaagagcct shuffledgctgttcaagacttctgcaggggtgaatatgtgcactctcatcgccatggacctgggc clonegagctgtgtgaggataccatgacctacaagtgtccacggatgaccgaggccgaacccg extendedacgatgtggattgctggtgcaatgcaactgatacttgggtgacctatgggacctgtag toccagacaggcgagcataggagggataagaggtccgtcgccctggaccctcacgttggt C15/fullctgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcaga prM/fulltccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgtt Ecctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcgtgggtaaactgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtgatggttcaggc a 202.6E12-D4 atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt RoundI caacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct shuffledgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc clonegagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg extendedaggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtac toccagtccggagagagaaggagggagaagaggtccgtcgccctgactcctcactctggt C15/fullatgggcctggaaaccagagccgagacatggatgagctctgagggagcttggaagcacg prM/fullcccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggctt Ecatggcttatatgattgggcagactggtattcagaggaccgtcttcttcgttctgatgatgctcgtggcaccatcttacggcatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaccaccaccatcttcgccggccacctgaagtgcaaggtgaggatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagcccccctttggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgca 203. 2/7-D1atgagatctgtgaccatgattctcatgctgctgcctactgctctggccttccatctga Round Icaacaagaggtggcgagcctaccctgatcgtgtccaagcaagaacgcggcaagagcct shuffledgctgttcaagacttctgcaggggtgaatatgtgcactctcatcgccatggacctgggc clonegagctgtgtgaggataccatgacctacaagtgtccacggatgaccgaggccgaacccg extendedacgatgtggattgctggtgcaatgcaactgatacttgggtgacctatgggacctgtag with DEN-1ccagacaggcgagcataggagggataagaggtccgtcgccctggaccctcacgttggt toctgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcaga C15/fulltccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgtt prM/fullcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctc Eatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatccagcctgggattttggctcaatcggaggggtgttcaccagcgtgggtaaactgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtgatggttcaggc a 204.2G11-D4 atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt RoundI caacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct shuffledgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc clonegagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg extendedaggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtac toccagtccggagagagaaggagggagaagaggtccgtcgccctgactcctcactctggt C15/fullatgggcctggaaaccagagccgagacatggatgagctctgagggagcttggaagcacg prM/fullcccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggctt Ecatggcttatatgattgggcagactggtattcagaggaccgtcttcttcgttctgatgatgctcgtggcaccatcttacggcatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcgtgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatgcgaggccgagcccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgca 205. 21C1atgaacaggaggaggagaactgtgggcgtgattatcatgctgatccctactgctatgg Shuffledccttccatctgacaacaagaaatggcgagcctcacatgatcgtgggcaggcaagagaa clonegggcaagagcctgctgttcaagactgaggacggcgtgaatatgtgcactctcatggcc (C15/fullatcgacctgggcgagctgtgtgaggataccgtgacctacaagtgtccactgctggtca prM/fullacaccgaacccgaggatatcgattgctggtgcaatctgacttctgcttgggtgaccta E)tgggacctgtaaccaggctggcgagcataggagggataagaggtccgtcgccctggctcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcacgcccagaggatcgagacctggattctgcgccatccaggctttatcatcatggctgccatcctggcttatacaattgggaccactcacttccagagagccctcatcttcattctgctcaccgccgtggcaccatctatgaccatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcggcgcctgggtggacctggtgctggagcacggcggctgcgtgaccaccatggcccagggcaagcccaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcggcgtggtgacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctggagtacaccgtggtggtgaccgtgcacaacggcgacacccacgccgtgggcaacgacaccagcaaccacggcgtgaccgccaccatcacccccaggagccccagcgtggaggtgaagctgcccgactacggcgagctgaccctggactgcgagcccaggagcggcatcgacttcaacgagatgatcctgatgaagatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagctcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaggaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtgc catggttcaggca206. 23C12 atgagatctgtgaccatgattctcatgctgctgcctactgctctggccttccatctgaShuffled caacaagaggtggcgagcctaccctgatcgtgtccaagcaagaacgcggcaagagcctclone gctgttcaagactgcttcagggatcaatatgtgcactctcatcgccatggacctgggc(C15/full gagatgtgtgacgataccgtgacctacaagtgtccacacatcaccgaggtcgaacccgprM/Full aggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtaa E)ccaggctggcgagcataggagggataagaggtccgtcgccctggttcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcacgcccagaggatcgagacctggattctgcgccatccaggctttatcatcatggctgccatcctggcttatacaattgggaccactcacttccagagagccctcatcttcattctgctcatgctcgtgacaccatctatgaccatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgcagaagaccgaggccacccagctggccaccctgaggaagctgtgcatcgagggcaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcaccaccgagatccagaacagcggcggcaccagcatcttcgccggccacctgaagtgcaggctgaagatggacaagctggagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgca 207. 23D5atgaaaactagcctctgcctgatgatgatgctgcctgctactctggccttccatctga Shuffledcatcaagagatggcgagcctaggatgatcgtgggcaagaacgaaagagggaagagcct clonegctgttcaagactgaggacggcgtgaatatgtgcactctcatcgccatggacctgggc (C15/fullgagctgtgtgaggataccatgacctacaagtgtccacggatgaccgaggtcgaacccg prM/fullaggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtaa E)ccaggctggcgagcataggagggataagaggtccgtcgccctggctcctcacgttggtatgggcctggacaccagaacccagacatggatgagcgctgagggagcttggaggcaggtcgagaaggttgaaacttgggctctgcgccatccaggctttacaatcctggccctgttcctggctcattacattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtcgagaaggttgaaacttgggctctgcgccatccaggctttacaatcctggccctgttcctggctcattacattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccaagatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccaccctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaacggcgacacccacgccgtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccagcaccagccaggagacctggaacaggcaggacctgctggtgaccttcaagaccgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcaccaccgagatccagaccagcggcaccaccaccatcttcgccggccacctgaagtgcaggctgaagatggacaagctgaccctgaagggcatgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccggcgccccctgcaaggtgcccatcgagatcagggacgtgaacaaggagaaggtggtgggcaggatcatcagcagcacccccctggccgagaacaccaacagcgtgaccaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcaccgccaggggcgccaggaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcgtgggtaaactgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtggtggttcaggca 208. 23F5atgagatctgccatcaccctgctctgcctgatccctactgttatggccttctctctgt Shuffledcaacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct clonegctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc (C15/fullgagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg prM/fullaggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtac E)ccagtccggagagagaaggagggagaagaggtccgtcgccctgactcctcactctggtatgggcctggacaccagaacccagacatggatgagcgctgagggagcttggaagcacgcccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggcttcatggcttatatgattgggcagactggtattcagaggaccgtcttcttcgttctgatgatgctcgtggcaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcggcgcctgggtggacctggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacacccgcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaggctgaagatggacaagctggagctgaagggcatgagctacagcatgtgcaccggcaagttccggatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcgtgggtaaactgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtgatggttcaggca 209. 23G3atgaaaactagcctctgcctgatgatgatgctgcctgctactctggccttccatctga Shuffledcaacaagaaatggcgagcctcacatgatcgtgggcaggcaagagaagggcaagagcct clonegctgttcaagacttctgcaggggtgaatatgtgcactctcatcgccatggacctgggc (C15/fullgagctgtgtgaggataccatgacctacaagtgtccacggatgaccgaggccgaacccg prM/fullaggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtac E)ccagtccggagagagaaggagggagaagaggtccgtcgccctgactcctcactctggtatgggcctggaaaccagagccgagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgcagaagaccgaggccacccagctggccaccctgaggaagctgtgcatcgagggcaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagaccagcggcaccaccaccatcttcgccggccacctgaagtgcaggctgaagatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcatcggcaaggccctgcaccaggttttcggtgcaatctatggcgcagccttttccggcgtctcttggaccatgaagatcctgatcggggtcatcatcacatggattggaatgaatagcaggagcacaagcctgagcgttagcctcgtcctggttggcgtcatcacactctacctgggtgccatggttcaggca 210. 23H7atgaaaactagcctctgcctgatgatgatgctgcctgctactctggccttccatctga Shuffledcatcaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct clonegctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc (C15/fullgagatgtgtgaggataccgtgacctacaagtgtccactgctgaggcagaacgaacccg prM/fullacgatgtggattgctggtgcaatgcaactgatacttgggtgacctatgggacctgtag E)ccagacaggcgagcataggagggataagaggtccgtcgccctggaccctcacgttggtctgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatgaccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccaagatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccaccctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccagcaccagccaggagacctggaacaggcaggacctgctggtgaccttcaagaccgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagaccagcggcaccaccaccatcttcaccggccacctgaagtgcaggctgaagatggacaagctgaccctgaagggcgtgagctacgtgacgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccgtggtgaccaagaaggaggagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcgtgggtaaactgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgccatggttcaggca 211. DEN-1atgggcatcatcttcatcctgctgatgctggtgaccccctctatggccatgaggtgcg PRM15/tEtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgt CO parentggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacPRM15/truncatedatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcg envelopeaggccaagatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccac codoncctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgtggacaggggc optimizedtggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttca parent)agtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccagcaccagccaggagacctggaacaggcaggacctgctggtgaccttcaagaccgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagaccagcggcaccaccaccatcttcgccggccacctgaagtgcaggctgaagatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacctgggcagcatcggcggcgtgttcaccagcgtgggcaag 212. DEN-2atgggcctgatcctgatcctgcagaccgccgtggccccctccatgacaatgaggtgca PRM15/tEtcggcatcagcaacagggacttcgtggagggcgtgagcggcggcagctgggtggacat CO parentcgtgctggagcacggctcctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgagcccagcctgaacgaggagcaggacaagaggttcgtgtgcaagcacagcatggtggacaggggctggggcaacggctgcggcctgttcggcaagggcggcatcgtgacctgcgccatgttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatcgtgctgctgcagatggaggacaaggcctggctggtgcacaggcagtggttcctggacctgcccctgccctggctgcccggcgccgacacccagggcagcaaccgtatccagaaggagaccctggtgaccttcaagaacccccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 213. DEN-3atggtggtgatcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgcg PRM15/tEtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt CO parentggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgcagaagaccgaggccacccagctggccaccctgaggaagctgtgcatcgagggcaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcaccaccgagatccagaacagcggcggcaccagcatcttcgccggccacctgaagtgcaggctgaagatggacaagctggagctgaagggcatgagctacgccatgtgcctgaacaccttcgtgctgaagaaggaggtgagcgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccgtggtgaccaagaaggaggagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaacagcctgggcaagatggtgcaccagatcttcggcagcgcctacacc 214. DEN-4atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcatgaggtgcg PRM15/tEtgggcgtgggcaacagggacttcgtggagggcgtgagcggcggcgcctgggtggacct CO parentggtgctggagcacggcggctgcgtgaccaccatggcccagggcaagcccaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcggcgtggtgacctgcgccaagttcagctgcagcggcaagatcaccggcaacctggtgcagatcgagaacctggagtacaccgtggtggtgaccgtgcacaacggcgacacccacgccgtgggcaacgacaccagcaaccacggcgtgaccgccaccatcacccccaggagccccagcgtggaggtgaagctgcccgactacggcgagctgaccctggactgcgagcccaggagcggcatcgacttcaacgagatgatcctgatgaagatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggagaagctgcgcatcaagggcatgagctacaccatgtgcagcggcaagttcagcatcgacaaggagatggccgagacccagcacggcaccaccgtggtgaaggtgaagtacgagggcaccggcgccccctgcaaggtgcccatcgagatcagggacgtgaacaaggagaaggtggtgggcaggatcatcagcagcacccccctggccgagaacaccaacagcgtgaccaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccatgtcc ggc 215.DEN-1 atgagatctgtgaccatgattctcatgctgctgcctactgctctggccttccatctga\C15/full caacaagaggtggcgagcctaccctgatcgtgtccaagcaagaacgcggcaagagcctprM/full gctgttcaagacttctgcaggggtgaatatgtgcactctcatcgccatggacctgggc E COgagctgtgtgaggataccatgacctacaagtgtccacggatgaccgaggccgaacccg parentacgatgtggattgctggtgcaatgcaactgatacttgggtgacctatgggacctgtag (whereinccagacaggcgagcataggagggataagaggtccgtcgccctggaccctcacgttggt a codonctgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcaga encodingtccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgtt a Metcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctc aminoatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcg acidtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgt residuegaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtg is alsoaccaaccccgccgtgctgaggaagctgtgcatcgaggccaagatcagcaacaccacca includedccgacagcaggtgccccacccagggcgaggccaccctggtggaggagcaggacaccaa as thecttcgtgtgcaggaggaccttcgtggacaggggctggggcaacggctgcggcctgttc firstggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggca codonagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcga contiguousccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccc andcaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgca prior togccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagag the firstctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgcc codon ofagcaccagccaggagacctggaacaggcaggacctgctggtgaccttcaagaccgccc theacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgc nucleotidecctgaccggcgccaccgagatccagaccagcggcaccaccaccatcttcgccggccac sequencectgaagtgcaggctgaagatggacaagctgaccctgaagggcgtgagctacgtgatgt encodinggcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgct C15)ggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatccagcctgggattttggctcaatcggaggggtgttcaccagcgtgggtaaactgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtgatggttcaggc a 216. DEN-2atgaacaggaggaggagaactgtgggcgtgattatcatgctgatccctactgctatgg \C15/fullccttccatctgacaacaagaaatggcgagcctcacatgatcgtgggcaggcaagagaa prM/fullgggcaagagcctgctgttcaagactgaggacggcgtgaatatgtgcactctcatggcc E COatcgacctgggcgagctgtgtgaggataccatcacctacaagtgtccactgctgaggc parentagaacgaacccgaggatatcgattgctggtgcaattcaacttctacttgggtgaccta (a codontgggacctgtaccaccacaggcgagcataggagggagaagaggtccgtcgccctggtt encodingcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggag a Metcttggaagcacgcccagaggatcgagacctggattctgcgccatccaggctttatcat residuecatggctgccatcctggcttatacaattgggaccactcacttccagagagccctcatc is alsottcattctgctcaccgccgtggcaccatctatgaccatgaggtgcatcggcatcagca includedacagggacttcgtggagggcgtgagcggcggcagctgggtggacatcgtgctggagca as thecggctcctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatc firstaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctga codonccaataccaccaccgagagcaggtgccccacccagggcgagcccagcctgaacgagga contiguousgcaggacaagaggttcgtgtgcaagcacagcatggtggacaggggctggggcaacggc andtgcggcctgttcggcaagggcggcatcgtgacctgcgccatgttcacctgcaagaaga prior toacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcacccc the firstccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatc codon ofaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtga theccatggagtgcagccccaggaccggcctggacttcaacgagatcgtgctgctgcagat nucleotideggaggacaaggcctggctggtgcacaggcagtggttcctggacctgcccctgccctgg sequencectgcccggcgccgacacccagggcagcaaccgtatccagaaggagaccctggtgacct encodingtcaagaacccccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgc C15)catgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaggttttcggtgcaatctatggcgcagccttttccggcgtctcttggaccatgaagatcctgatcggggtcatcatcacatggattggaatgaatagcaggagcacaagcctgagcgttagcctcgtcctggttggcgtcatcacactctacctgggtgc catggttcaggca217. DEN-3 atgaaaactagcctctgcctgatgatgatgctgcctgctactctggccttccatctga\C15/full catcaagagatggcgagcctaggatgatcgtgggcaagaacgaaagagggaagagcctprM/full gctgttcaagactgcttcagggatcaatatgtgcactctcatcgccatggacctgggc E COgagatgtgtgacgataccgtgacctacaagtgtccacacatcaccgaggtcgaacccg parentaggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtaa is codonccaggctggcgagcataggagggataagaggtccgtcgccctggctcctcacgttggt encodingatgggcctggacaccagaacccagacatggatgagcgctgagggagcttggaggcagg a Mettcgagaaggttgaaacttgggctctgcgccatccaggctttacaatcctggccctgtt aminocctggctcattacattgggacctctctgactcagaaggtcgtcatcttcattctgctc acidatgctcgtgacaccatctatgaccatgaggtgcgtgggcgtgggcaacagggacttcg residuetggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgt is alsogaccaccatggccaagaacaagcccaccctggacatcgagctgcagaagaccgaggcc includedacccagctggccaccctgaggaagctgtgcatcgagggcaagatcaccaacatcacca as theccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaa firstctacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttc codonggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggca contiguousaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcga andccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggcc prior toagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagcccca the firstggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggat codon ofggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccacc thegagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgcca nucleotideagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgac sequencecggcaccaccgagatccagaacagcggcggcaccagcatcttcgccggccacctgaag encodingtgcaggctgaagatggacaagctggagctgaagggcatgagctacgccatgtgcctga C15)acaccttcgtgctgaagaaggaggtgagcgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccgtggtgaccaagaaggaggagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaacagcctgggcaagatggtgcaccagatcttcggttcagcatataccgcactgttttccggcgtctcttggatcatgaagatcggtatcggggtcctcctcacatggattggactgaatagcaagaatacaagcatgagctttagctgcatcgctattggcatcatcacactctacctgggtgtggtggttcaggca 218. DEN-4atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt \C15/fullcaacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct prM/fullgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc E COgagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg parentaggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtac (a codonccagtccggagagagaaggagggagaagaggtccgtcgccctgactcctcactctggt encodingatgggcctggaaaccagagccgagacatggatgagctctgagggagcttggaagcacg Met aminocccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggctt acidcatggcttatatgattgggcagactggtattcagaggaccgtcttcttcgttctgatg residueatgctcgtggcaccatcttacggcatgaggtgcgtgggcgtgggcaacagggacttcg is alsotggagggcgtgagcggcggcgcctgggtggacctggtgctggagcacggcggctgcgt includedgaccaccatggcccagggcaagcccaccctggacttcgagctgatcaagaccaccgcc as theaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcacca firstccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagca codongtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttc contiquousggcaagggcggcgtggtgacctgcgccaagttcagctgcagcggcaagatcaccggca andacctggtgcagatcgagaacctggagtacaccgtggtggtgaccgtgcacaacggcga prior tocacccacgccgtgggcaacgacaccagcaaccacggcgtgaccgccaccatcaccccc the firstaggagccccagcgtggaggtgaagctgcccgactacggcgagctgaccctggactgcg codon ofagcccaggagcggcatcgacttcaacgagatgatcctgatgaagatgaagaagaagac thectggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgcc nucleotidegacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgcccc sequenceacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgc encodingctgaagtgcaaggtgaggatggagaagctgcgcatcaagggcatgagctacaccatgt C15)gcagcggcaagttcagcatcgacaaggagatggccgagacccagcacggcaccaccgtggtgaaggtgaagtacgagggcaccggcgccccctgcaaggtgcccatcgagatcagggacgtgaacaaggagaaggtggtgggcaggatcatcagcagcacccccctggccgagaacaccaacagcgtgaccaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgc a 219. DEN-1atgagatctgtgaccatgattctcatgctgctgcctactgctctggccttccatctga with restcaacaagaggtggcgagcctaccctgatcgtgtccaagcaagaacgcggcaagagcct of COgctgttcaagacttctgcaggggtgaatatgtgcactctcatcgccatggacctgggc PRM-encodinggagctgtgtgaggataccatgacctacaagtgtccacggatgaccgaggccgaacccg sequenceacgatgtggattgctggtgcaatgcaactgatacttgggtgacctatgggacctgtagccagacaggcgagcataggagggataagaggtccgtcgccctggaccctcacgttggtctgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcatgcaattgggacctctattactcag 220. DEN-2atgaacaggaggaggagaactgtgggcgtgattatcatgctgatccctactgctatgg with restccttccatctgacaacaagaaatggcgagcctcacatgatcgtgggcaggcaagagaa of COgggcaagagcctgctgttcaagactgaggacggcgtgaatatgtgcactctcatggccC15/PRM-encodingatcgacctgggcgagctgtgtgaggataccatcacctacaagtgtccactgctgaggc sequenceagaacgaacccgaggatatcgattgctggtgcaattcaacttctacttgggtgacctatgggacctgtaccaccacaggcgagcataggagggagaagaggtccgtcgccctggttcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcacgcccagaggatcgagacctggattctgcgccatccaggctttatcat 221. DEN-3atgaaaactagcctctgcctgatgatgatgctgcctgctactctggccttccatctga with restcatcaagagatggcgagcctaggatgatcgtgggcaagaacgaaagagggaagagcct of COgctgttcaagactgcttcagggatcaatatgtgcactctcatcgccatggacctgggcC15/PRM-encodinggagatgtgtgacgataccgtgacctacaagtgtccacacatcaccgaggtcgaacccg sequenceaggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtaaccaggctggcgagcataggagggataagaggtccgtcgccctggctcctcacgttggtatgggcctggacaccagaacccagacatggatgagcgctgagggagcttggaggcaggtcgagaaggttgaaacttgggctctgcgccatccaggctttacaatcctggccctgttcctggctcattacattgggacctctctgactcag 222. DEN-4atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt with restcaacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct of COgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc C15/PRMgagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg encodingaggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtac sequenceccagtccggagagagaaggagggagaagaggtccgtcgccctgactcctcactctggtatgggcctggaaaccagagccgagacatggatgagctctgagggagcttggaagcacgcccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggcttcatggcttatatgattgggcagactggtattcag 223. DEN-1ctgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttgga with restccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaggagcac of COaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtgatg Env-encodinggttcaggca sequence 224. DEN-2gccttttccggcgtctcttggaccatgaagatcctgatcggggtcatcatcacatgga with restttggaatgaatagcaggagcacaagcctgagcgttagcctcgtcctggttggcgtcat of COcacactctacctgggtgccatggttcaggca Env-encoding sequence 225. DEN-3gcactgttttccggcgtctcttggatcatgaagatcggtatcggggtcctcctcacat with restggattggactgaatagcaagaatacaagcatgagctttagctgcatcgctattggcat of COcatcacactctacctgggtgtggtggttcaggca Env-encoding sequence 226. DEN-4ggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaacca with restatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactctt of COcctgggtttcaccgttcacgca Env-encoding seq 227. DEN-1MRSVTMILMLLPTALAFHLTTRGGEPTLIVSKQERGKSLLFKTSAGVNMCTLIAMDLG \C15/fullELCEDTMTYKCPRMTEAEPDDVDCWCNATDTWVTYGTCSQTGEHRRDKRSVALDPHVG prM/fullLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILL E parentMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGMGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIPGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQA 228. DEN-2MNRRRRTVGVIIMLIPTAMAFHLTTRNGEPHMIVGRQEKGKSLLFKTEDGVNMCTLMA \C15/fullIDLGELCEDTITYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCTTTGEHRREKRSVALV prM/fullPHVGMGLETRTETWMSSEGAWKHAQRIETWILRHPGFIIMAAILAYTIGTTHFQRALI E parentFILLTAVAPSMTMRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEATQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEIVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNRIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVSLVLVGVITLYLGAMVQA 229. DEN-3MKTSLCLMMMLPATLAFHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLG \C15/fullEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVG prM/fullMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSLTQKVVIFILL E parentMLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGQGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWIMKIGIGVLLTWIGLNSKNTSMSFSCIAIGIITLYLGVVVQA 230. DEN-4MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG \C15/fullEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSG prM/fullMGLETRAETWMSSEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQRTVFFVLM E parentMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELIKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTATITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA 231. DEN-1actgtcttctttgtcctaatgatgctggtcgccccatcctacggaatgcgatgcgtag PRM15/Egagtaggaaacagagactttgtggaaggagtctcaggtggagcatgggtcgacctggt trunc WTgctagaacatggaggatgcgtcacaaccatggcccagggaaaaccaaccttggatttt cDNAgaactgactaagacaacagccaaggaagtggctctgttaagaacctattgcattgaag sequencecctcaatatcaaacataactacggcaacaagatgtccaacgcaaggagagccttatct fragmentgaaagaggaacaggaccaacagtacatttgccggagagatgtggtagacagagggtgg Nucleicggcaatggctgtggcttgtttggaaaaggaggagttgtgacatgtgcgaagttttcat acidsgttcggggaagataacaggcaatttggtccaaattgagaaccttgaatacacagtggt 894-2285tgtaacagtccacaatggagacacccatgcagtaggaaatgacacatccaatcatgga ofgttacagccatgataactcccaggtcaccatcggtggaagtcaaattgccggactatg GenBankgagaactaacactcgattgtgaacccaggtctggaattgactttaatgagatgattct Acc. No.gatgaaaatgaaaaagaaaacatggctcgtgcataagcaatggtttttggatctgcct AB074761cttccatggacagcaggagcagacacatcagaggttcactggaattacaaagagagaatggtgacatttaaggttcctcatgccaagagacaggatgtgacagtgctgggatctcaggaaggagccatgcattctgccctcgctggagccacagaagtggactccggtgatggaaatcacatgtttgcaggacatcttaagtgcaaagtccgtatggagaaattgagaatcaagggaatgtcatacacgatgtgttcaggaaagttttcaattgacaaagagatggcagaaacacagcatgggacaacagtggtgaaagtcaagtatgaaggtgctggagctccgtgtaaagtccccatagagataagagatgtaaacaaggaaaaagtggttgggcgtatcatctcatccacccctttggctgagaataccaacagtgtaaccaacatagaattagaacccccctttggggacagctacatagtgataggtgttggaaacagcgcattaacactccattggttcaggaaagggagttccattggcaagatgtttgagtccacatacagaggtgcaaaacgaatggccattctaggtgaaacagcttgggattttggttccgttggtggactgttcacatcattgggaaaggctgtgcaccaggtttttggaagtgtgtatacaaccatgtttgga 232. DEN-2gtcctgatattcatcctactgacagccatcgctccttcaatgacaatgcgctgcatag PRM15/Egaatatcaaatagggactttgtggaaggagtgtcaggagggagttgggttgacatagt trunc WTtttagaacatggaagttgtgtgacgacgatggcaaaaaataaaccaacactggacttt cDNAgaactgataaaaacagaagccaaacaacccgccaccttaaggaagtactgtatagagg sequencectaaactgaccaacacgacaacagactcgcgctgcccaacacaaggggaacccaccct fragmentgaatgaagagcaggacaaaaggtttgtctgcaaacattccatggtagacagaggatggggaaatggatgtggattatttggaaaaggaggcatcgtgacctgtgccatgttcacat Nucleicgcaaaaagaacatggagggaaaaattgtgcagccagaaaacctggaatacactgtcgt acidstataacacctcattcaggggaagaacatgcagtcggaaatgacacaggaaaacatggt 892-2274aaagaagtcaagataacaccacagagctccatcacagaggcggaactgacaggctatg ofgcactgttacgatggagtgctctccaagaacgggcctcgacttcaatgagatggtgtt GenBankgctgcaaatgaaagacaaagcttggctggtgcacagacaatggttcctagacctaccg Acc. No.ttgccatggctgcccggagcagacacacaaggatcaaattggatacagaaagagacac NC_001474tggtcaccttcaaaaatccccatgcgaaaaaacaggatgttgttgtcttaggatcccaagagggggccatgcatacagcactcacaggggctacggaaatccagatgtcatcaggaaacctgctgttcacaggacatcttaagtgcaggctgagaatggacaaattacaacttaaagggatgtcatactccatgtgcacaggaaagtttaaagttgtgaaggaaatagcagaaacacaacatggaacaatagtcattagagtacaatatgaaggagacggctctccatgcaagaccccttttgagataatggatctggaaaaaagacatgttttgggccgcctgaccacagtcaacccaattgtaacagaaaaggacagtccagtcaacatagaagcagaacctccattcggagacagctacatcatcataggagtggaaccaggacaattgaagctggactggttcaagaaaggaagttccatcggccaaatgtttgagacaacaatgaggggagcgaaaagaatggccattttgggcgacacagcctgggattttggatctctgggaggagtgttcacatcaataggaaaggctctccaccaggtttttggagcaatctacggggct 233. DEN-3gttatttttatactattaatgctggttaccccatccatgacaatgagatgtgtgggag PRM15/Etaggaaacagagattttgtggaaggcctatcgggagctacgtgggttgacgtggtgct trunc WTcgagcacggtgggtgtgtgactaccatggctaagaacaagcccacgctggacatagag cDNActtcagaagactgaggccactcagctggcgaccctaaggaagctatgcattgagggaa sequenceaaattaccaacataacaaccgactcaagatgtcccacccaaggggaagcgattttacc fragmenttgaggagcaggaccagaactacgtgtgtaagcatacatacgtggacagaggctggggaaacggttgtggtttgtttggcaagggaagcttggtgacatgcgcgaaatttcaatgtt Nucleictagaatcaatagagggaaaagtggtgcaacatgagaacctcaaatacaccgtcatcat acidscacagtgcacacaggagaccaacaccaggtgggaaatgaaacgcagggagttacggct (nt) 893-2263gagataacatcccaggcatcaaccgctgaagccattttacctgaatatggaaccctcg ofggctagaatgctcaccacggacaggtttggatttcaatgaaatgattttattgacaat GenBankgaagaacaaagcatggatggtacatagacaatggttctttgacttacccctaccatgg Acc. No.acatcaggagctacaacaaaaacaccaacttggaacaggaaagagcttcttgtgacat M25277ttaaaaatgcacatgcaaaaaagcaagaagtagttgtccttggatcacaagagggagcaatgcatacagcactgacaggagctacagagatccaaacctcaggaggcacaagtatttttgcggggcacttaaaatgtagactcaagatggacaaattgaaactcaaggggatgagctatgcaatgtgcttgaatacctttgtgttgaagaaagaagtctccgaaacgcagcatgggacaatactcattaaggttgagtacaaaggggaacatgcaccctgcaagattcctttctccacggaggatggacaagggaaagctcacaatggcagactgatcacagccaatccagtggtgaccaagaaggaggagcctgtcaacattgaggctgaacctccttttggggaaagtaatatagtaattggaagtggagacaaagccctgaaaatcaactggtacaggaagggaagctcgattgggaagatgttcgaggccactgccagaggtgcaaggcgcatggccatcttgggagacacagcctgggactttggatcagtgggtggtgttttgaattcattagggaaaatggtccaccaaatatttgggagtgcttacaca 234. DEN-4actgtcttctttgtcctaatgatgctggtcgccccatcctacggaatgcgatgcgtag PRM15/Egagtaggaaacagagactttgtggaaggagtctcaggtggagcatgggtcgacctggt trunc WTgctagaacatggaggatgcgtcacaaccatggcccagggaaaaccaaccttggatttt cDNAgaactgactaagacaacagccaaggaagtggctctgttaagaacctattgcattgaag sequencecctcaatatcaaacataactacggcaacaagatgtccaacgcaaggagagccttatct fragmentgaaagaggaacaggaccaacagtacatttgccggagagatgtggtagacagagggtggggcaatggctgtggcttgtttggaaaaggaggagttgtgacatgtgcgaagttttcat Nucleicgttcggggaagataacaggcaatttggtccaaattgagaaccttgaatacacagtggt acidstgtaacagtccacaatggagacacccatgcagtaggaaatgacacatccaatcatgga 894-2285gttacagccatgataactcccaggtcaccatcggtggaagtcaaattgccggactatg ofgagaactaacactcgattgtgaacccaggtctggaattgactttaatgagatgattct GenBankgatgaaaatgaaaaagaaaacatggctcgtgcataagcaatggtttttggatctgcct Acc. No.cttccatggacagcaggagcagacacatcagaggttcactggaattacaaagagagaa M14931tggtgacatttaaggttcctcatgccaagagacaggatgtgacagtgctgggatctcaggaaggagccatgcattctgccctcgctggagccacagaagtggactccggtgatggaaatcacatgtttgcaggacatcttaagtgcaaagtccgtatggagaaattgagaatcaagggaatgtcatacacgatgtgttcaggaaagttttcaattgacaaagagatggcagaaacacagcatgggacaacagtggtgaaagtcaagtatgaaggtgctggagctccgtgtaaagtccccatagagataagagatgtaaacaaggaaaaagtggttgggcgtatcatctcatccacccctttggctgagaataccaacagtgtaaccaacatagaattagaacccccctttggggacagctacatagtgataggtgttggaaacagcgcattaacactccattggttcaggaaagggagttccattggcaagatgtttgagtccacatacagaggtgcaaaacgaatggccattctaggtgaaacagcttgggattttggttccgttggtggactgttcacatcattgggaaaggctgtgcaccaggtttttggaagtgtgtatacaaccatgtttggaatggtggtgatcttcatcctgctgatgctggtgaccccctccatgacaatgaggtgcg 235. 18H6tgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgt checkggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggac thisttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcg PRM15/tEaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccat Round IIcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggc shuffledtggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttca clonecctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcgccatctacggcgcc 236. 25B6MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG (C15/fullELCEDTITYKCPLLRQDEPEDIDCWCNATDTWVTYGTCNQAGEHRRDKRSVALTPHSG lengthMGLETRAETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILL prM/fullMLVTPSMAMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEA length E)TQLATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQQYICRRDVVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLKMDKLELKGMSYAMCLNTFVLKKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGESYIVIGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSMSFSCIAIGIITLYLGVVVQA 237. 25B10MRSVTMILMLLPTTLAFHLTSRDGEPRMIVAKHERGRPLLFKTTEGINKCTLIAMDLG (C15/fullEMCDDTVTYKCPHITEVEPEDIDCWCNSTSTWVTYGTCNQAGEHRRDKRSVALAPHVG lengthMGLDTRTQTWMSAEGAWKHAQRIETWILRHPGFIIMAAILAYTIGTTHFQRALIFILL prM/fullTAAAPSMTMRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDIELLKTEV length E)TNPAVLRKLCIEAKISNTTTDSRCPTQGEATLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKTAHAKKQEVVVLGSQEGAMHSALAGATEVQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGKSNIVIGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSIGKALHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRSTSLSMTCIAVGMVTLYLGVMVQA 238. 25D4MRSVTMILMLLPTALAFHLTTRGGEPTLIVSKQERGKSLLFKTSAGINMCTLIAMDLG (C15/fullEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALVPHVG lengthMGLETRTETWMSSEGAWKHAQRIETWILRHPGFTILALFLAHYIGTSLTQKVVIFILL prM/fullMLVTPSMTMRCIGISNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFELIKTEA length E)TQPATLRKYCIEAKLTNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVVVTVHNGDTHAVGNDTSNHGVTATITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHTALTGATEIQMSSGNHMFAGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITANPVVTKKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVQA 239. 25E11MKTSLCLMMMLPATLAFHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLG (C15/fullEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCTQSGERRREKRSVALTPHSG lengthMGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILL prM/fullMLVTPSMAMRCVGVGNRDFVEGLSGATWVDIVLEHGSCVTTMAKNKPTLDFELIKTEA length E)TQPATLRKLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGALTLDCSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGGTSIFAGHLKCRLKMDKLTLKGVSYVMCTGSFKLEKEVSETQHGTILIKVEYKGEDAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQA 240. 25H4MRSVTMILMLLPTALAFHLTTRGGEPLMIVAKHERGKSLLFKTASGINMCTLIAMDLG (C15/fullEMCDDTVTYKCPRMTEVEPEDIDCWCNLTSTWVTYGTCTTTGEHRREKRSVALVPHVG lengthMGLETRTETWMSSEGAWKQIQKVETWALRHPGFTILALFLAHYIGTSLTQEVVIFILL prM/fullMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEV length E)TNPAVLRKLCIEAKISNTTTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIVTVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVSLVLVGVITLYLGAMVQA 241. 27A11MKTSLCLMMMLPTALAFHLTTRGGEPTLIVSKQERGKSLLFKTSAGVNMCTLIAMDLG (C15/fullELCEDTMTYKCPRMTEAEPDDVDCWCNATDTWVTYGTCSQTGEHRREKRSVALVPHVG lengthMGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILL prM/fullMLVTPSMAMRCVGIGNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDIELLKTEV length E)TNPAVLRKLCIEAKISNTTTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPDYGELTLDCEPRSGIDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGGTSIFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA 242. 27G6MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG (C15/fullEMCEDTVTYKCPLLVNTEPEDIDCWCNSTSTWVMYGTCSQTGEHRRDKRSVALVPHVG lengthMGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHYIGTSLTQKVVIFILL prM/fullMLVTPSMTMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEV length E)TNPAVLRKLCIEAKISNTTTDSRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLEYTIVITPHSGEEHAVGNDTSNHGVTATITPTTETPTWNRKELLVTFKNAHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGVGDSALTLHWFRKGSSIGKMFESTYRGARRMAILGDTAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA 243. 28A11MRSTITLLCLIPTVMAFHLTTRNGEPRMIVGKNERGKSLLFKTEDGVNMCTLMAIDLG (C15/fullELCRDTITYKCPLLVNTEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVG lengthMGLETRTETWMSSEGAWKQIQKVETWALRHPGFTILALFLAHAIGTSITQKGIIFILL prM/fullMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEV length E)TNPAVLRKLCIEAKITNITTDSRCPTQGEAILPEEQDQNYVCKHSMVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILMKMKKKTWLVHRQWFLDLPLPWTSGASTSQETWNRQDLLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAIGIITLYLGVVVQA 244. 28C1MKTSLCLMMMLPATMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG (C15/fullELCEDTMTYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCNQAGEHRRDKRSVALDPHVG lengthLGLETRTQTWMSAEGAWRQVEKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILL prM/fullMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTA length E)KEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTATITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRM*KLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGIGDKALKINWFRKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA 245. 28D11MRSTITLLCLIPTVMAFSLSTRDGEPLMIVGKNERGKSLLFKTASGINMCTLIAMDLG (C15/fullEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQAGEHRRDKRSVALAPHVG lengthMGLDTRTQTWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILL prM/fullMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELQKTEA length E)TQLATLRKLCIEGKISNTTTDSRCPTQGEATLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGVITLYLGAMVQA 246. 28E12MKTSLCLMMMLPATLAFHLTSRDGEPRMIVGKNERGKSLLFKTTEGINKCTLIAMDLG (C15/fullEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVTYGTCTTTGEHRREKRSVALDPHVG lengthLGLETRTETWMSSEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSLTQKVVIFILL prM/fullMLVTPSMTMRCVGAGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDFELIKTEA length E)TQPATLRKYCIEAKITNITTDSRCPTQGEAILNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGVVTCAMFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVRGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVGDSALTLHWFRKGSSIGKMFESTYRGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGMNSRSTSLSVSLVLVGVITLYLGAMVQA 247. 28F9MNRRRRTVGVIIMLIPTAMAFHLTTRNGEPRMIVAKHERGRPLLFKTTEGINKCTLIA (C15/fullMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVTYGTCSQTGEHRREKRSVALD lengthPHVGLGLETRTETWMNSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGII prM/fullFILLMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAKNKPTLDFELI length E)KTEATQPATLRKYCIEAKLTNTTTDSRCPTQGEPSLKEEQDQQYICRRDVVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTSNHGVTATITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFKKGSSIGKMFEATARGARRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA 248. 28H3MRPTITLLCIMMMLPATLAFHLTTRNGEPHMIVGRQEKGKSLLFKTEDGVNMCTLMAT (C15/fullDLGELCEDTITYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCTTTGEHRREKRSVALVP lengthHVGMGLETRTETWMSSEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQRTVFF prM/fullVLMMLVAPSYGMRCVGVGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLK length E)TEVTNPAVLRKYCIEAKLTNTTTESRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYTMCSGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVIDKEKPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVSLVLVGVITLYLGAMVQA 249. 28H9MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG (C15/fullEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALVPHVG lengthMGLETRTETWMSAEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSLTQKVVIFILL prM/fullMLVTPSMTMRCVGVGNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEA length E)TQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHTALTGTTEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWTMKILIGVIITWIGMNSRSTSLSVSLVLVGVITLYLGAMVQA 250. 16G11-D4MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG (16G11EMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSG extendedMGLETRAETWMSSEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQRTVFFVLM toMLVAPSYGMRCVGVGNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDIELLKTEV C15/fullTNPAVLRKLCIEASISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLF lengthGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQA prM/STVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWLVHKQWFLDLPLPWTAGADT fullSEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLK length ECRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDG with WTQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGK Den-4 EMFEATARGARRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRIL proteinIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA residues) 251. 16G11-MRSVTMILMLLPTTLAFHLTSRDGEPRMIVAKHERGRPLLFKTTEGINKCTLIAMDLG 25B10extEMCDDTVTYKCPHITEVEPEDIDCWCNSTSTWVTYGTCNQAGEHRRDKRSVALAPHVG (16G11MGLDTRTQTWMSAEGAWKHAQRIETWILRHPGFIIMAAILAYTIGTTHFQRALIFILL extendedTAAAPSMTMRCIGISNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDIELLKTEV toTNPAVLRKLCIEASISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLF C15/fullGKGSVVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQA lengthSTVEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWLVHKQWFLDLPLPWTAGADT prM/SEVHWNHKERMVTFKTAHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLK fullCRLRMDKLQLKGMSYVMCTGKFQIVKEIAETQHGTIVIRVQYEGDGAPCKIPFSTEDG length EQGKAHNGRLITANPIVIDKEKPVNIELEPPFGDSYIVVGAGDKALKINWYKKGSSIGK withMFEATARGARRMAILGDTAWDFGSIGGVFTSIGKALHQVFGSVYTTMFGGVSWMVRIL 25B10IGFLVLWIGTNSRSTSLSMTCIAVGMVTLYLGVMVQA residues of E protein) 252.18H5-D4 MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLG (18H6EMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSG extendedMGLETRAETWMSSEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQRTVFFVLM toMLVAPSYGMRCVGVGNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTA C15/fullKEVALLRTYCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLF lengthGKGSLVTCAKFTCKKNMEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQA prM/STVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADT fullSEVHWNHKERMVTFKNAHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLK length ECRLKMDKLQLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTEDG with WTQGKAHNGRLITVNPIVIDKEKPVNIELEPPFGESYIVVGAGEKALKLSWFKKGSSIGK Den-4 EMFEATARGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRIL proteinIGFLVLWTGTNSRNTSMAMSCIAVGGITLFLGFTVHA residues) 253. 18H6-MRSVTMILMLLPTTLAFHLTSRDGEPRMIVAKHERGRPLLFKTTEGINKCTLIAMDLG 25B10extEMCDDTVTYKCPHITEVEPEDIDCWCNSTSTWVTYGTCNQAGEHRRDKRSVALAPHVG (18H6MGLDTRTQTWMSAEGAWKHAQRIETWILRHPGFIIMAAILAYTIGTTHFQRALIFILL extendedTAAAPSMTMRCIGISNRDFVEGVSGATWVDVVLEHGGCVTTMAKNKPTLDFELIKTTA toKEVALLRTYCIEASISNITTATRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLF C15/fullGKGSLVTCAKFTCKKNMEGNIVQPENLEYTIVITPHTGDQHQVGNDTQGVTVEITPQA lengthSTVEAILPEYGTLGLECSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTAGADT prM/SEVHWNHKERMVTFKNAHAKRQDVTVLGSQEGAMHSALAGATEIQMSSGNLLFTGHLK fullCRLKMDKLQLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTEDG length EQGKAHNGRLITVNPIVIDKEKPVNIELEPPFGESYIVVGAGEKALKLSWFKKGSSIGK withMFEATARGAKRMAILGDTAWDFGSIGGVFTSIGKALHQVFGSVYTTMFGGVSWMVRIL 25B10IGFLVLWIGTNSRSTSLSMTCIAVGMVTLYLGVMVQA residues of E protein)254. >16G11-D4atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt (16G11caacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct extendedgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc togagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg C15/fullaggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtac lengthccagtccggagagagaaggagggagaagaggtccgtcgccctgactcctcactctggt prM/atgggcctggaaaccagagccgagacatggatgagctctgagggagcttggaagcacg fullcccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggctt length Ecatggcttatatgattgggcagactggtattcagaggaccgtcttcttcgttctgatg with WTatgctcgtggcaccatcttacggcatgaggtgcgtgggcgtgggcaacagggacttcg Den-4 Etggagggcgtgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgt proteingaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtg residues)accaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccaccctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgca 255. >16G11-atgagatctgtgaccatgattctcatgctgctgcctactactctggccttccatctga 25B10extcatcaagagatggcgagcctaggatgatcgtggccaagcacgaaagagggaggcctct (16G11gctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc extendedgagatgtgtgacgataccgtgacctacaagtgtccacacatcaccgaggtcgaacccg toaggatatcgattgctggtgcaattcaacttctacttgggtgacctatgggacctgtaa C15/fullccaggctggcgagcataggagggataagaggtccgtcgccctggctcctcacgttggt lengthatgggcctggacaccagaacccagacatggatgagcgctgagggagcttggaagcacg prM/cccagaggatcgagacctggattctgcgccatccaggctttatcatcatggctgccat fullcctggcttatacaattgggaccactcacttccagagagccctcatcttcattctgctc length Eaccgccgcggcaccatctatgaccatgaggtgcatcggcatcagcaacagggacttcg withtggagggcgtgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgt 25B10 Egaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtg proteinaccaaccccgccgtgctgaggaagctgtgcatcgaggccagcatcagcaacaccacca residues)ccgacagcaggtgccccacccagggcgaggccaccctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcatcggcaaggccctgcaccaggttttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtgatggttcaggca 256. >18H6-D4atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt (18H6caacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct extendedgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc togagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg C15/fullaggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtac lengthccagtccggagagagaaggagggagaagaggtccgtcgccctgactcctcactctggt prM/atgggcctggaaaccagagccgagacatggatgagctctgagggagcttggaagcacg fullcccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggctt length Ecatggcttatatgattgggcagactggtattcagaggaccgtcttcttcgttctgatg with WTatgctcgtggcaccatcttacggcatgaggtgcgtgggcgtgggcaacagggacttcg Den-4 Etggagggcgtgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgt proteingaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccaccgcc residues)aaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgca 257. >18H6-atgagatctgtgaccatgattctcatgctgctgcctactactctggccttccatctga 25B10extcatcaagagatggcgagcctaggatgatcgtggccaagcacgaaagagggaggcctct (18H6gctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc extendedgagatgtgtgacgataccgtgacctacaagtgtccacacatcaccgaggtcgaacccg toaggatatcgattgctggtgcaattcaacttctacttgggtgacctatgggacctgtaa C15/fullccaggctggcgagcataggagggataagaggtccgtcgccctggctcctcacgttggt lengthatgggcctggacaccagaacccagacatggatgagcgctgagggagcttggaagcacg prM/cccagaggatcgagacctggattctgcgccatccaggctttatcatcatggctgccat fullcctggcttatacaattgggaccactcacttccagagagccctcatcttcattctgctc length Eaccgccgcggcaccatctatgaccatgaggtgcatcggcatcagcaacagggacttcg withtggagggcgtgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgt 25B10gaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccaccgcc residuesaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcacca of Eccgccaccaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaa protein)ctacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaagaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcatcggcaaggccctgcaccaggttttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtgatggttcaggca 258. >25B6atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt (C15/fullcaacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct lengthgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc prM/fullgagctgtgtgaggataccatcacctacaagtgtccactgctgaggcaggacgaacccg length E)aggatatcgattgctggtgcaatgcaactgatacttgggtgacctatgggacctgtaaccaggctggcgagcataggagggataagaggtccgtcgccctgactcctcactctggtatgggcctggaaaccagagccgagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgcagaagaccgaggccacccagctggccaccctgaggaagctgtgcatcgagggcaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccagcaccagccaggagacctggaacaggcaggacctgctggtgaccttcaagaccgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctggagctgaagggcatgagctacgccatgtgcctgaacaccttcgtgctgaagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcgtgggtaaactgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaggagcacaagcatgagctttagctgcatcgctattggcatcatcacactctacctgggtgtggtggttcaggca 259. >25B10atgagatctgtgaccatgattctcatgctgctgcctactactctggccttccatctga (C15/fullcatcaagagatggcgagcctaggatgatcgtggccaagcacgaaagagggaggcctct lengthgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc prM/fullgagatgtgtgacgataccgtgacctacaagtgtccacacatcaccgaggtcgaacccg length E)aggatatcgattgctggtgcaattcaacttctacttgggtgacctatgggacctgtaaccaggctggcgagcataggagggataagaggtccgtcgccctggctcctcacgttggtatgggcctggacaccagaacccagacatggatgagcgctgagggagcttggaagcacgcccagaggatcgagacctggattctgcgccatccaggctttatcatcatggctgccatcctggcttatacaattgggaccactcacttccagagagccctcatcttcattctgctcaccgccgcggcaccatctatgaccatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcggcagctgggtggacatcgtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccaagatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccaccctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccacccagggcaaggcccacaacggcaggctgatcaccgccaaccccgtggtgaccaagaaggaggagcccgtgaacatcgaggccgagccccccttcggcaagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcatcggcaaggccctgcaccaggttttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtgatggttcaggca 260. >25D4atgagatctgtgaccatgattctcatgctgctgcctactgctctggccttccatctga (C15/fullcaacaagaggtggcgagcctaccctgatcgtgtccaagcaagaacgcggcaagagcct lengthgctgttcaagacttctgcagggatcaatatgtgcactctcatcgccatggacctgggc prM/fullgagatgtgtgacgataccgtgacctacaagtgtccacacatcaccgaggtcgaacccg length E)aggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtaaccaggctggcgagcataggagggataagaggtccgtcgccctggttcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcacgcccagaggatcgagacctggattctgcgccatccaggctttacaatcctggccctgttcctggctcattacattgggacctctctgactcagaaggtcgtcatcttcattctgctcatgctcgtgacaccatctatgaccatgaggtgcatcggcatcagcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtggtggtgaccgtgcacaacggcgacacccacgccgtgggcaacgacaccagcaaccacggcgtgaccgccaccatcacccccaggagccccagcgtggaggtgaagctgcccgactacggcgagctgaccctggactgcgagcccaggagcggcatcgacttcaacgagatgatcctgatgaagatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaaccacatgttcgccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgccaaccccgtggtgaccaagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcaggc a 261. >25E11atgaaaactagcctctgcctgatgatgatgctgcctgctactctggccttccatctga (C15/fullcatcaagagatggcgagcctaggatgatcgtgggcaagaacgaaagagggaagagcct lengthgctgttcaagactgcttcagggatcaatatgtgcactctcatcgccatggacctgggc prM/fullgagatgtgtgacgataccgtgacctacaagtgtccacacatcaccgaagtcgaacccg length E)aggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtacccagtccggagagagaaggagggagaagaggtccgtcgccctgactcctcactctggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacatcgtgctggagcacggctcctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagctgtgcatcgagggcaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccagcaccagccaggagacctggaacaggcaggacctgctggtgaccttcaagaccgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcaccaccgagatccagaacagcggcggcaccagcatcttcgccggccacctgaagtgcaggctgaagatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtgagcgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaaggtgcccatcgagatcagggacgtgaacaaggagaaggtggtgggcaggatcatcagcagcacccccctggccgagaacaccaacagcgtgaccaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcgtgggtaaactgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaggagcacaagcctgagcatgacctgcatcgctgttggcatggtcacactctacctgggtgtgatggttcaggca 262. >25H4atgagatctgtgaccatgattctcatgctgctgcctactgctctggccttccatctga (C15/fullcaacaagaggtggcgagcctctcatgatcgtggccaagcacgaaagagggaagagcct lengthgctgttcaagactgcttcagggatcaatatgtgcactctcatcgccatggacctgggc prM/fullgagatgtgtgacgataccgtgacctacaagtgtccacggatgaccgaggtcgaacccg length E)aggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtaccaccacaggcgagcataggagggagaagaggtccgtcgccctggttcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacaatcctggccctgttcctggctcattacattgggacctctctgactcaggaggtcgtcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccaagatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccagcaccagccaggagacctggaacaggcaggacctgctggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaggttttcggtgcaatctatggcgcagccttttccggcgtctcttggaccatgaagatcctgatcggggtcatcatcacatggattggaatgaatagcaggagcacaagcctgagcgttagcctcgtcctggttggcgtcatcacactctacctgggtgccatggttcaggca 263. >27Allatgaaaactagcctctgcctgatgatgatgctgcctactgctctggccttccatctga (C15/fullcaacaagaggtggcgagcctaccctgatcgtgtccaagcaagaacgcggcaagagcct lengthgctgttcaagacttctgcaggggtgaatatgtgcactctcatcgccatggacctgggc prM/fullgagctgtgtgaggataccatgacctacaagtgtccacggatgaccgaggccgaacccg length E)acgatgtggattgctggtgcaatgcaactgatacttgggtgacctatgggacctgtagccagacaggcgagcataggagggagaagaggtccgtcgccctggttcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcgtgagcggcggcagctgggtggacatcgtgctggagcacggctcctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccaagatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgactacggcgagctgaccctggactgcgagcccaggagcggcatcgacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcaccaccgagatccagaacagcggcggcaccagcatcttcgccggccacctgaagtgcaaggtgaggatggagaagctgcgcatcaagggcatgagctacaccatgtgcagcggcaagttcagcatcgacaaggagatggccgagacccagcacggcaccaccgtggtgaaggtgaagtacgagggcaccggcgccccctgcaaggtgcccatcgagatcagggacgtgaacaaggagaaggtggtgggcaggatcatcagcagcacccccctggccgagaacaccaacagcgtgaccaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgca 264. >27G6atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt (C15/fullcaacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct lengthgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc prM/fullgagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg length E)aggatatcgattgctggtgcaattcaacttctacttgggtgatgtatgggacctgtagccagacaggcgagcataggagggataagaggtccgtcgccctggttcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcattacattgggacctctctgactcagaaggtcgtcatcttcattctgctcatgctcgtgacaccatctatgaccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccaagatcagcaacaccaccaccgacagcaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacaccagcaaccacggcgtgaccgccaccatcacccccaggagccccagcgtggaggtgaagctgcccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggagaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccgtggtgaccaagaaggaggagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgc a 265. >28A11atgagatctaccatcaccctgctctgcctgatccctactgttatggccttccatctga (C15/fullcaacaagaaatggcgagcctaggatgatcgtgggcaagaacgaaagagggaagagcct lengthgctgttcaagactgaggacggcgtgaatatgtgcactctcatggccatcgacctgggc prM/fullgagctgtgtgaggataccatcacctacaagtgtccactgctggtcaacaccgaacccg length E)aggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtaaccaggctggcgagcataggagggataagaggtccgtcgccctggctcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacaatcctggccctgttcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacagcatggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgatgaagatgaagaagaagacctggctggtgcacaggcagtggttcctggacctgcccctgccctggaccagcggcgccagcaccagccaggagacctggaacaggcaggacctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagaccagcggcatccaccaccacttcgccggccacctgaagtgcaaggtgaggatggagaagctgcgcatcaagggcatgagctacaccatgtgcagcggcaagttcagcatcgacaaggagatggccgagacccagcacggcaccaccgtggtgaaggtgaagtacgagggcaccggcgccccctgcaaggtgcccatcgagatcagggacgtgaacaaggagaaggtggtgggcaggatcatcagcagcacccccctggccgagaacaccaacagcgtgaccaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctattggcatcatcacactctacctgggtgtggtggttcaggca 266. >28C1atgaaaactagcctctgcctgatgatgatgctgcctgctactatggccttctctctgt (C15/fullcaacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctct lengthgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc prM/fullgagctgtgtgaggataccatgacctacaagtgtccactgctgaggcagaacgaacccg length E)aggatatcgattgctggtgcaattcaacttctacttgggtgacctatgggacctgtaaccaggctggcgagcataggagggataagaggtccgtcgccctggaccctcacgttgttctgggcctggaaaccagaacccagacatggatgagcgctgagggagcttggaggcaggtcgagaaggttgaaacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacctactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagggcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcggcgtggtgacctgcgccaagttcagctgcagcggcaagatcaccggcaacctggtgcagatcgagaacctggagtacaccgtggtggtgaccgtgcacaacggcgacacccacgccgtgggcaacgacaccagcaaccacggcgtgaccgccaccatcacccccaggagccccagcgtggaggtgaagctgcccgactacggcgagctgaccctggactgcgagcccaggagcggcatcgacttcaacgagatgatcctgatgaagatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatgtagaagctgcgcatcaagggcatgagctacaccatgtgcagcggcaagttcagcatcgacaaggagatggccgagacccagcacggcaccaccgtggtgaaggtgaagtacgagggcaccggcgccccctgcaaggtgcccatcgagatcagggacgtgaacaaggagaaggtggtgggcaggatcatcagcagcacccccctggccgagaacaccaacagcgtgaccaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcatcggcgacaaggccctgaagatcaactggttcaggaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgc a 267. >28D11atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt (C15/fullcaacaagagatggcgagcctctcatgatcgtgggcaagaacgaaagagggaagagcct lengthgctgttcaagactgcttcagggatcaatatgtgcactctcatcgccatggacctgggc prM/fullgagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg length E)aggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtacccaggctggcgagcataggagggataagaggtccgtcgccctggctcctcacgttggtatgggcctggacaccagaacccagacatggatgagctctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgcagaagaccgaggccacccagctggccaccctgaggaagctgtgcatcgagggcaagatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccaccctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagaccagcggcaccaccaccatcttcgccggccacctgaagtgcaggctgaagatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccgtggtgaccaagaaggaggagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcgtcatcacactctacctgggtgccatggttcaggca 268. >28E12atgaaaactagcctctgcctgatgatgatgctgcctgctactctggccttccatctga (C15/fullcatcaagagatggcgagcctaggatgatcgtgggcaagaacgaaagagggaagagcct lengthgctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggc prM/fullgagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccg length E)aggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtaccaccacaggcgagcataggagggagaagaggtccgtcgccctggaccctcacgttggtctgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaggcaggtcgagaaggttgaaacttgggctctgcgccatccaggctttacaatcctggccctgttcctggctcattacattgggacctctctgactcagaaggtcgtcatcttcattctgctcatgctcgtgacaccatctatgaccatgaggtgcgtgggcgcgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccatcctgaacgaggagcaggacaagaggttcgtgtgcaagcacagcatggtggacaggggctggggcaacggctgcggcctgttcggcaagggcggcgtggtgacctgcgccatgttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagaatcacccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgcggggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaggaggatggccatcctcggcgatacagcctgggattttggctcaatcggaggggtgttcaccagcgtgggtaaactgattcatcagattttcggtacagcatatggcgtgctgttttccggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggaatgaatagcaggagcacaagcctgagcgttagcctcgtcctggttggcgtcatcacactctacctgggtgccatggttcaggca 269. >28F9atgaacaggaggaggagaactgtgggcgtgattatcatgctgatccctactgctatgg (C15/fullccttccatctgacaacaagaaatggcgagcctcggatgatcgtggccaagcacgaaag lengthagggaggcctctgctgttcaagactactgaagggatcaataagtgcactctcatcgcc prM/fullatggacctgggcgagatgtgtgaggataccgtgacctacaagtgtccactgctggtca length E)acaccgaacccgaggatatcgattgctggtgcaatctgacttctacttgggtgacctatgggacctgtagccagacaggcgagcataggagggagaagaggtccgtcgccctggaccctcacgttggtctgggcctggaaaccagaaccgagacatggatgaactctgagggagcttggaagcagatccagaaggttgagacttgggctctgcgccatccaggctttacagtcattgccctgttcctggctcatgcaattgggacctctattactcagaagggcatcatcttcattctgctcatgctcgtggcaccatcttacggcatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcgtgagcggcggcgcctgggtggacctggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgacagcaggtgccccacccagggcgagcccagcctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggcaccatcacccccaggagccccagcgtggaggtgaagctgcccgactacggcgagctgaccctggactgcgagcccaggagcggcatcgacttcaacgagatgatcctgatgaagatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtaccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggagaagctgcgcatcaagggcatgagctacaccatgtgcagcggcaagttcagcatcgacaaggagatggccgagacccagcacggcaccaccgtggtgaaggtgaagtacgagggcaccggcgccccctgcaaggtgcccatcgagatcagggacgtgaacaaggagaaggtggtgggcaggatcatcagcagcacccccctggccgagaacaccaacagcgtgaccaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggttt caccgttcacgca270. >28H3 atgagacctaccatcaccctgctctgcctgatgatgatgctgcctgctactctggcct(C15/full tccatctgacaacaagaaatggcgagcctcacatgatcgtgggcaggcaagagaaggglength caagagcctgctgttcaagactgaggacggcgtgaatatgtgcactctcatggccatcprM/full gacctgggcgagctgtgtgaggataccatcacctacaagtgtccactgctgaggcagalength E) acgaacccgaggatatcgattgctggtgcaattcaacttctacttgggtgacctatgggacctgtaccaccacaggcgagcataggagggagaagaggtccgtcgccctggttcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcacgcccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggcttcatggcttatatgattgggcagactggtattcagaggaccgtcttcttcgttctgatgatgctcgtggcaccatcttacggcatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacaccatgtgcagcggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaggttttcggtgcaatctatggcgcagccttttccggcgtctcttggaccatgaagatcctgatcggggtcatcatcacatggattggaatgaatagcaggagcacaagcctgagcgttagcctcgtcctggttggcgtcatcacactctacctgggtgccatggttca ggca271. >28H9 atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgt(C15/full caacaagagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctctlength gctgttcaagactactgaagggatcaataagtgcactctcatcgccatggacctgggcprM/full gagatgtgtgaggataccgtgacctacaagtgtccactgctggtcaacaccgaacccglength E) aggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtacccagtccggagagagaaggagggagaagaggtccgtcgccctggttcctcacgttggtatgggcctggaaaccagaaccgagacatggatgagcgctgagggagcttggaggcaggtcgagaaggttgaaacttgggctctgcgccatccaggctttacaatcctggccctgttcctggctcattacattgggacctctctgactcagaaggtcgtcatcttcattctgctcatgctcgtgacaccatctatgaccatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcgtgagcggcggcagctgggtggacatcgtgctggagcacggctcctgcgtgaccaccatggccaagaacaagcccaccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagggcgagcccagcctgaacgaggagcaggacaagaggttcgtgtgcaagcacagcatggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacaccgccctgaccggcaccaccgagatccagaacagcggcggcaccagcatcttcgccggccacctgaagtgcaggctgaagatggacaagctggagctgaagggcatgagctacgccatgtgcctgaacaccttcgtgctgaagaaggaggtgagcgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccgtggtgaccaagaaggaggagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgctgaacagcctgggcaagatggtgcaccagatcttcggttcagcatataccgcactgttttccggcgtctcttggaccatgaagatcctgatcggggtcatcatcacatggattggaatgaatagcaggagcacaagcctgagcgttagcctcgtcctggttggcgtcatcacactctacctgggtgccatggttcaggca 272. 6C6 PRM15atgaccgtgttcttcatcctgatgatgctggtgaccccctctatggcc DNA 273. 6F4 PRM15atggccgtgttcttcatcctgctgatgctggtgaccccctccatgaca DNA 274. 7A9 PRM15atgaccgtgttcttcatcctgatgatgctggtggccccctcctacgcc DNA 275. 11E2Atgaccgtgttcttcatcctgctgatgctggtggccccctcctacggc PRM15 DNA 276. 12E3atggccgtgttcttcatcctgatgatgctggtggccccctcctacggc PRM15 DNA 277. 15C2atggtggtgatcttcatcctgctgatgctggtgaccccctctatggcc PRM15 DNA 278. 17A12atgaccgtgttcttcatcctgatgatgctggtgaccccctccatgaca PRM15 DNA 279. 17D5atgaccgtgttcttcatcctgctgatgctggtgaccccctccatgaca PRM15 DNA 280. 17G5atgaccgtgttcttcatcctgatgatgctggtggccccctctatggcc PRM15 DNA 281. DEN-1atgggcatcatcttcatcctgctgatgctggtgaccccctctatggcc PRM15 of DEN-1 PRM15/tECO parent 282. DEN-2 atgggcctgatcctgatcctgcagaccgccgtggccccctccatgacaPRM15 of DEN-2 PRM15/tE CO parent 283. DEN-3atggtggtgatcttcatcctgctgatgctggtgaccccctccatgaca PRM15 of DEN-3 PRM15/tECO parent 284. DEN-4 atgaccgtgttcttcatcctgatgatgctggtggccccctcctacggcPRM15 of DEN-4 PRM15/tE CO parent 285. 2/7-NPRMatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct Round Igggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag clone notactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacgg cgcc 286.2G11-NPRM atgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctRound I gggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no tactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccat gttcggc 287.5/21 NPRM atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctRound I gggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagccshuffled caccctggacatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagclone tactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagg no PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacgg cgcc 288.6C6-NPRM atgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcgtgagcggcggcgcctRound I gggtggacctggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no tactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcggcatcgtgacctgcgccatgttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgatgaagatgaagaacaaggcctggatggtgcacaggcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcactgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgcct acacc 289.6E12-NPRM atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctRound I gggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagccshuffled caccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagclone no ctgtgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaccaccaccatcttcgccggccacctgaagtgcaaggtgaggatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagcccccctttggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccac catgttcggc290. 6F4-NPRM atgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcgtgagcggcggcgcctRound I gggtggacctggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagclone no tactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggagaagctgcgcatcaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 7A9-NPRMatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcgtgagcggcgccacct Round Igggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacatcgagctgcagaagaccgaggccacccagctggccaccctgaggaag clone noctgtgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccaccctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagagcaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcaccaccgagatccagaacagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccac catgttcggc292. 11E2-NPRMatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaag clone noctgtgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctactgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgagaccctgcactggttcaggaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacgg cgcc 293.12E3-NPRM atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctRound II gggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagccshuffled caccctggacatcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no tactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacac caccatgttcggc294. 13E2-NPRMatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcgtgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacc clone notactgcatcgaggccagcatcagcaacattcaccaccgccaccaggtgcccacccagg PRMgcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacac caccatgttcggc295. 14E9-NPRMatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcgtgagcggcggcgcct Round IIgggtggacctggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag clone notactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgatgaagatgaagaacaaggcctggatggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgcctacacc 296. 13E11-atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcgtgagcggcggcgcct NPRMgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag shuffledtactgcatcgaggccagcatcagcaacatcaccaccgacaccaggtgccccacccagg clone nogtgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagagcaagacctggctagtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacgg cgcc 297.16E8-NPRM atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctRound II gggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no tactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 298. 16E10-atgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct NPRMgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaag shuffledtactgcatcgaggccaaaatcagcaacaccaccaccgacagcaggtgccccacccagg clone nogcgaggccaccctggtggaggagcaggaccagaactacgtgtgcaagcacacctacgt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcgccctgggcctggagtgcagccccaggaccggcctggacttcaacgagagcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaacagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccat gttcggc 299.18E9-NPRM atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctRound II gggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaagclone no tactgcatcgaggccaagctgaccaataccaccaccgccaccaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagagcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaacagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccat gttcggc 300.18E10- atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacct NPRMgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacatcgagctgcagaagaccgaggccacccagctggccaccctgaggaag shuffledtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagg clone nogcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacac caccatgttcggc301. 18E11- atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctNPRM gggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacttcgagctgatcaagaccaccgtcaaggaggtggccctgctgaggacc shuffledtactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg clone nogcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 302. 11B1-NPRMatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag clone notactgcatcgaggccaagctgaccaataccaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgatacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgtagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaggaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcgccatctacggcgcc 303. 11B8-NPRMatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaag clone notactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacaggggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggagaagctgtcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccat gttcggc 304.11C4-NPRM atgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctRound II gggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagccshuffled caccctggacatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaccclone no tactgcatcgaggccagcatcagcaacatcaccaccgagagcaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagagcaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcagcccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtgggcgacaaggccctgaagctgcactggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgcct acacc 305.11C11- atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacct NPRMgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaag shuffledctgtgcatcgaggccagcatcagcaacatcaccaccgagagcaggtgccccacccagg clone nogcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcgtggtgggcgccggcgagaaggccctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 306. 12H4-NPRMatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag clone notactgcatcgaggccagcatcagcaacatcaccaccgacagcaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgtcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggtgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgcctacacc 307. 13F11-atgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcgtgagcggcggcgcct NPRMgggtggacctggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag shuffledtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagg clone nogcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacgagaagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacgg cgcc 308.14B1-NPRM atgaggtgcgtgggcatcagcaacagggacttcgtggagggcgtgagcggcggcgcctRound II gggtggacctggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagclone no tactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctgaagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgcctacacc 309. 14G10-atgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct NPRMgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaag shuffledtactgcatcgaggccaagatcaccaacatcaccaccgacaccaggtgccccacccagg clone nogcgaggccatcctgcccgaggagcaggaccagcagtacatctgcaggagggacgtggt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttcacctgcaagaagaacatggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacgg cgcc 310.14H2-NPRM atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcgtgagcggcggcgcctRound II gggtggacctggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no tactgcatcgaggccaagatcaccaacatcaccaccgccaccaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacaagggcgaggacgccccctgcaagatccccttcagcagccaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 311. 15C2-NPRMatgaggtgcgtcggcatcagcaacagggacttcgtggagggcctgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaag clone notactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagg PRMgcgagccctacctgaaggaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccat gttcggc 312.15D4-NPRM atgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctRound II gggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no tactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcctggagcccatcgagggcaaggtggtgcagcccgagaacctggagtacaccatcgtgatcaccccccacagcggcgaggagcacgccgtgggcaacgacactggcaagcacggcaaggagatcaagatcaccccccagagcagcatcaccgaggccgagctgaccggctacggcaccgtgaccatggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaagtgttcggcgccatctacgg cgcc 313.15H4-NPRM atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctRound II gggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccgaggtgaccaaccccgccaccctgaggaagclone no tactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccaacctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacaggggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccgcctgaagtgcaggctgaagatggacaagctgtcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagcccccctttggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaacagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccat gttcggc 314.16B4-NPRM atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacctRound II gggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagccshuffled caccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagclone no ctgtgcatcgaggccagcatcagcaacatcaccaccgacagcaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagagcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaccaccaccatcttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccac catgttcggc315. 16F12- atgaggtgcgtgggcgtgggcaacggggacttcgtggagggcctgagcggcgccacctNPRM gggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacttcgagctgatcaagaccatcgccaaggaggtggccctgctgaggacc shuffledtactgcatcgaggccagcatcagcaacatcaccaccgacagcaggtgccccacccagg clone nogcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccagcggcgccaccaccgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcaccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaccaccaccatcttcgccggccacctgaagtgcaaggtgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccctggagatcatggacctggagaagaggcacgtgctgggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 316. 16G11-atgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacct NPRMgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaag shuffledctgtgcatcgaggccagcatcagcaacaccaccaccgacagcaggtgccccacccagg clone nogcgaggccaccctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcctgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccat gttcggc 317.17A12- atgaggtgcgtgggcgtgggcaacagggacttcgtggagggcctgagcggcgccacct NPRMgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacttcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag shuffledtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagg clone nogcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcctggagcccatcgagggcaaggtggtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacgagatgatcctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctgaccggcgccaccgagatccagaccagcggcaccaccaccatcttcgccggccacctgaagtgcagggtgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccgtcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacctcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 318. 17D5-NPRMatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaggaacaagcc shuffledcaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag clone notactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagcagtacatctgcaggagggacgtggtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccagcggcgccaccaccgagacccccacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 319. 17D11-atgaggtgcgtgggcaccggcaaccgcgacttcgtggagggcctgagcggcgccacct NPRMgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggcccagggcaagcc Round IIcaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag shuffledtactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagg clone nogcgagccctacctgaaggaggagcaggaccagaactacgtgtgcaagcacacctacgt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacaccatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 320. 17F5-NPRMatgaggtgcgtgggcaccggcaaccgcgacttcgtggagggcctgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggcccagggcaagcc shuffledcaccctggacatcgagctgatcaagaccgaggccacccagcccgccaccctgaggaag cone notactgcatcgaggccaagctgaccaataccaccaccgagagcaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcgtggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacgacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcctgggcaaggccgtgcaccagatcttcggcagcgtgtacaccaccat gttcggc 321.16F11- atgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct NPRMgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc Round IIcaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacc shuffledtactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg clone nogcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgt PRMggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggctcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctagtgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggctcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctaggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgtcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgcactggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccat gttcggc 322.17G5-NPRM atgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctRound II gggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no tactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggctcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctagtgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggctcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctaggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcgtgttcaccagcatcggcaaggccctgcaccaggtgttcggcgccatctacggcgcc 323. 17H3-NPRMatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacc clone notactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggctcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctaggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtgatcggcgtgggcgacagcgccctgaccctgagctggttcaggaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaag 324. 17H10-NPRMatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacc clone notactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggctcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctaggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcgacggcaaccacatgttcgccggccacctgaagtgcaaggtgaggatggacaagctgcagctgaagggcatgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcgtgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacaccaccat gttcggc 325.17H12-NPRM atgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctRound II gggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no tactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacac caccatgttcggc326. 18A9-NPRMatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacc clone notactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggctcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctaggcagccaggagggcgccatgcacagcgccctggccggcgccaccgaggtggacagcggcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgaccctgaagggcgtgagctacgtgatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgcagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaccctgcactggttcaggaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgttcaccagcgt gggcaag 327.18B7-NPRM atgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctRound II gggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no tactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagccccccttcggcgacagctacatcgtggtgggcgccggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccaccat gttcggc 328.18D7-NPRM atgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacctRound II gggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagccshuffled caccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggaccclone no ctgtgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttcaagtgcgtgaccaagctggagggcaagatcgtgcagtacgagaacctgaagtacagcgtgatcgtgaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacgagatgatcctgctgaccatgaagaacaaggcctggatggtgcacaggcagtggttcttcgacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaccgcccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgagatccagatgagcagcggcaccaccaccatcttcgccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcctgatcaaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggccagatgttcgagagcacctacaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggczgcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccac catgttcggc329. 18H2-NPRMatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacc clone notactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacaccagcaaccacggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaaggtgccccacgccaagaagcaggacgtggtggtgctgggcagccaggagggcgccatgcacaccgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgacggcgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgccaaccccgtggtgaccaagaaggaggagcccgtgaacatcgaggccgagccccccttcggcgagagcaacatcgtgatcggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgagaccaccatgaggggcgccaagaggatggccatcttgggcgacaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaagatggtgcaccagatcttcggcagcgtgtacac caccatgttcggc330. 18H6-NPRMatgaggtgcatcggcatcagcaacagggacttcgtggagggcgtgagcggcgccacct Round IIgggtggacgtggtgctggagcacggcggctgcgtgaccaccatggccaagaacaagcc shuffledcaccctggacttcgagctgatcaagaccaccgccaaggaggtggccctgctgaggacc clone notactgcatcgaggccagcatcagcaacatcaccaccgccaccaggtgccccacccagg PRMgcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctgatcacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgacacccagggcgtgaccgtggagatcaccccccaggccagcaccgtggaggccatcctgcccgagtacggcaccctgggcctggagtgcagccccaggactggcctggacttcaacagggtggtgctgctgaccatgaagaagaagagctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccgacaccagcgaggtgcactggaaccacaaggagaggatggtgaccttcaagaacgcccacgccaagaggcaggacgtgaccgtgctgggcagccaggagggcgccatgcacagcgccctggccggcgccaccgagatccagatgagcagcggcaacctgctgttcaccggccacctgaagtgcaggctgaagatggacaagctgcagctgaagggcgtgagctacgtgatgtgcaccggcagcttcaagctggagaaggaggtggccgagacccagcacggcaccgtgctggtgcaggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcaccgaggacggccagggcaaggcccacaacggcaggctgatcaccgtgaaccccatcgtgatcgacaaggagaagcccgtgaacatcgagctggagccccccttcggcgagagctacatcgtggtgggcgccggcgagaaggccctgaagctgagctggttcaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaagaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcgccatctacggcgcc 331. pMaxVaxacacatagcgccggcgctagctgagcaaaaggccagcaaaaggcca (“pMV” or “PMV”) primer 1332. pMaxVax aactctgtgagacaacagtcataaatgtacagatatcagaccaagtttactcatatatprimer 2 ac 333. pMaxVax ggcttctcacagagtggcgcgccgtgtctcaaaatctct primer3 334. pMaxVax ttgctcagctagcgccggcgccgtcccgtcaagtcagcgt primer 4 335.pMaxVax agatctgtttaaaccgctgatcagcctcgactgtgccttc primer 5 336. pMaxVaxacctctaaccactctgtgagaagccatagagcccaccgca primer 6 337. pMaxVaxggatccggtacctctagagaattcggcggccgcagatctgtttaaaccgctga primer 7 338.DEN-1 E- MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKtruncated LCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCparent AKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGRIATITPQAPTSEIQ (tE)LTDYGALTLDCSPRTGLDFNRVVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDLGSIGGVFTSVGK 339. DEN-2 E-MRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEATQPATLRK truncatedYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTC parentAMFTCKKNMEGNIVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAE (tE)LTGYGTVTMECSPRTGLDFNEIVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNRIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVQYEGDGSPCKIPLEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGA 340. DEN-3 E-MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRK truncatedLCIEGKITNITTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTC parentAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNDTQGVTVEITPQASTVEAILP (tE)EYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWTSGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYT 341. DEN-4 E-MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELIKTTAKEVALLRT truncatedYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTC parentAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTATITPRSPSVEVK (tE)LPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNHKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGDSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFG 342. 30F10atgagatctaccatcaccctgctctgcctgatccctactgttatggccttctctctgtcaacaa(C15/fullgagatggcgagcctctcatgatcgtggccaagcacgaaagagggaggcctctgctgttcaagacprM/fulltactgaagggatcaataagtgcactctcatcgccatggacctgggcgagatgtgtgaggatacc E)gtgacctacaagtgtccactgctggtcaacaccgaacccgaggatatcgattgctggtgcaatctgacttctacttgggtgatgtatgggacctgtacccagtccggagagagaaggagggagaagaggtccgtcgccctggctcctcacgttggtatgggcctggacaccagaactcagacatggatgagcgctgagggagcttggaagcacgcccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggcttcatggcttatatgattgggcagactggtattcagaggaccgtcttcttcgttctgatgatgctcgtggcaccatcttacggcatgaggtgcgtgggcgtgggcaacagggacttcgtggagggcgtgagcggcggcgcctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccaccctgaggaagtactgcatcgaggccaagatcaccaacatcaccaccgacagcaggtgccccacccagggcgaggccaccctggtggaggagcaggacaccaacttcgtgtgcaggaggaccttcgtggacaggggctggggcaacggctgcggcctgttcggcaagggcggcatcgtgacctgcgccaagttcaagtgcgtgaccaagctggagggcaacatcgtgcagcccgagaacctggagtacaccatcgtgatcaccccccacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcaccaccgagatccagaacagcggcggcaccctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcaaggtggagtacaagggcgaggacgccccctgcaagatccccttcagcaccgaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgaccgagaaggacagccccgtgaacatcgaggccgagccccccttcggcgagagctacatcgtggtgggcatcggcgacaaggccctgaagatcaactggtacaagaagggcagcagcatcggcaagatgttcgaggccaccgccaggggcgccaggaggatggccatcctgggcgagaccgcctgggacttcggcagcgtgggcggcctgctgaccagcctgggcaaggccgtgcaccaggtgttcggcagcgtgtacaccacaatgtttggcggcgtctcttggatggtgagaatcctgatcgggttcctcgtcctgtggattggaaccaatagcaggaatacaagcatggccatgagctgcatcgctgttggcggcatcacactcttcctgggtttcaccgttcacgca 343. 30F10MRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEM (C15/fullCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALAPHVGM prM/fullGLDTRTQTWMSAEGAWKHAQRVESWILRNPRFALLAGFMAYMIGTGIQRTVFFVL E)MMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPATLRKYCIEAKITNITTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGGIVTCAKFKCVTKLEGNIVQPENLEYTIVITPHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGTTEIQNSGGTLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIKVEYKGEDAPCKIPFSTEDEKGVTQNGRLITANPIVTEKDSPVNIEAEPPFGESYIVVGIGDKALKINWYKKGSSIGKMFEATARGARRMAILGETAWDFGSVGGLLTSLGKAVHQVFGSVYTTMFGGVSWMVRILIGFLVLWIGTNSRNTSMAMSCIAVGGITLFLGFTVHA 344. 30G2atgagatctgtgaccatgattctcatgctgctgcctactgctctggccttccatctgacaacaa(C15/fullgagatggcgagcctaggatgatcgtgggcaagaacgaaagagggaagagcctgctgttcaagacprM/fulltgcttcagggatcaatatgtgcactctcatcgccatggacctgggcgagatgtgtgaggatacc E)atgacctacaagtgtccacggatgaccgaggccgaacccgacgatgtggattgctggtgcaatgcaactgatacttgggtgacctatgggacctgtagccagacaggcgagcataggagggataagaggtccgtcgccctggaccctcacgttggtctgggcctggaaaccagaaccgagacatggatgagctctgagggagcttggaagcacgcccagagggttgaatcttggattctgcgcaatccacgctttgcactcctggctggcttcatggcttatatgattgggcagactggtattcagaggaccgtcatcttcattctgctcatgctcgtgacaccatctatggccatgaggtgcgtgggcatcggcaaccgcgacttcgtggagggcctgagcggcgccacctgggtggacgtggtgctggagcacggcagctgcgtgaccaccatggccaagaacaagcccaccctggacatcgagctgctgaagaccgaggtgaccaaccccgccgtgctgaggaagctgtgcatcgaggccaagatcagcaacaccaccaccgacagcaggtgccccacccagggcgaggccatcctgcccgaggagcaggaccagaactacgtgtgcaagcacacctacgtggacaggggctggggcaacggctgcggcctgttcggcaagggcagcctggtgacctgcgccaagttccagtgcctggagcccatcgagggcaaggtggtgcagcacgagaacctgaagtacaccgtgatcatcaccgtgcacaccggcgaccagcaccaggtgggcaacgagaccaccgagcacggcaccatcgccaccatcaccccccaggcccccaccagcgagatccagctgaccgactacggcgccctgaccctggactgcagccccaggaccggcctggacttcaacagggtggtgctgctgaccatgaagaagaagacctggctggtgcacaagcagtggttcctggacctgcccctgccctggaccgccggcgccagcaccagccaggagacctggaacaggaaggagctgctggtgaccttcaagaacgcccacgccaagaagcaggaggtggtggtgctgggcagccaggagggcgccatgcacaccgccctgaccggcgccaccgaggtggacagcggcgacggcaacctgctgttcaccggccacctgaagtgcaggctgaggatggacaagctgcagctgaagggcatgagctacagcatgtgcaccggcaagttccagatcgtgaaggagatcgccgagacccagcacggcaccatcgtgatcagggtgaagtacgagggcaccgacgccccctgcaagatccccttcagcagccaggacgagaagggcgtgacccagaacggcaggctgatcaccgccaaccccatcgtgatcgacaaggagaagcccgtgaacatcgaggccgagcccccctttggcgacagctacatcatcatcggcgtggagcccggccagctgaagctgcactggttcaagaagggcagcagcatcggccagatgttcgagaccaccatgaggggcgccaagaggatggccatcctgggcgacaccgcctgggacttcggcagcctgggcggcgtgttcaccagcatcggcaaggccctgcaccaggttttcggtgcaatctatggcgtgctgtttttcggcgtctcttggaccatgaagatcggtattggcatcctcctcacatggctgggactgaatagcaagaatacaagcatgagctttagctgcatcgctattggcatcatcacactctacctgggtgtggtggttcaggca 345. 30G2MRSVTMILMLLPTALAFHLTTRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLG (C15/fullEMCEDTMTYKCPRMTEAEPDDVDCWCNATDTWVTYGTCSQTGEHRRDKRSVALDPH prM/fullVGLGLETRTETWMSSEGAWKHAQRVESWILRNPRFALLAGFMAYMIGQTGIQRTVIFI E)LLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKNKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQHENLKYTVIITVHTGDQHQVGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNRVVLLTMKKKTWLVHKQWFLDLPLPWTAGASTSQETWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEVDSGDGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFQIVKEIAETQHGTIVIRVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVIDKEKPVNIEAEPPFGDSYIIIGVEPGQLKLHWFKKGSSIGQMFETTMRGAKRMAILGDTAQDFGSLGGVFTSIGKALHQVFGAIYGVLFFGVSWTMKIGIGILLTWLGLNSKNTSMSFSCIAIGIITLYLGVVVQA

[0797] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims. Forexample, all the techniques and apparatus described above may be used invarious combinations.

[0798] All references, including publications, patent applications,patents, and/or other documents cited herein, including those notspecifically indicated as being incorporated by reference when citedabove, are each hereby incorporated by reference in their entirety forall purposes to the same extent as if each such reference wereindividually and specifically indicated to be incorporated by referenceherein in its entirety for all purposes and were set forth in itsentirety herein.

[0799] The use of the terms “a” and “an” and “the” and similar referentsin the context of describing the invention (especially in the context ofthe following claims) are to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext. Terms such as “including,” “having,” “comprising,”“containing,” and the like are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwiseindicated, and as encompassing the phrases “consisting of” and“consisting essentially of.” Recitation of ranges of values herein aremerely intended to serve as a shorthand method of referring individuallyto each separate value of the range, unless otherwise indicated herein,and each separate value is incorporated into the specification as if itwere individually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention. Theheadings provided in the description of the invention are includedmerely for convenience and are not intended to be limiting in the scopeof the disclosure.

[0800] The citation of any patent or patent document herein does notreflect any view concerning the patentability of the subject matterdescribed or claimed in such patent documents. Rather, such patentdocuments may be cited merely to provide convenient reference forsuitable techniques and compositions, including techniques andcompositions otherwise well known in the art.

[0801] All amino acid or nucleotide sequences of one of theaforementioned sequence patterns are to be considered individuallydisclosed herein. Thus, for example, an amino acid sequence pattern ofthree residues, where a “Xaa” represents one of the amino acid positionsin the pattern represents a disclosure of twenty different amino acidsequences (i.e., one sequence for each naturally occurring amino acidresidue that could be present in the Xaa position).

[0802] Any of the techniques and any of the characteristics of the viralvector particle compositions of the invention can be combined in anysuitable manner, unless otherwise stated or clearly contradicted bycontext.

[0803] Preferred embodiments and aspects of this invention are describedherein, including the best mode known to the inventors for carrying outthe invention. Variations of those preferred embodiments may becomeapparent to those of ordinary skill in the art upon reading theforegoing description. The inventors expect skilled artisans to employsuch variations as appropriate, and the inventors intend for theinvention to be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040009469). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

What is claimed is:
 1. A recombinant or synthetic polypeptide comprisingan amino acid sequence that has at least about 90% amino acid sequenceidentity to at least one amino acid sequence selected from the group ofSEQ ID NOS:66, 69, 89, 93, 108, and
 110. 2. The recombinant or syntheticpolypeptide of claim 1, wherein the recombinant or synthetic polypeptideinduces an immune response in a subject against at least one flavivirus.3. The recombinant or synthetic polypeptide of claim 1, wherein therecombinant or synthetic polypeptide induces an immune response in asubject against at least one dengue virus.
 4. The recombinant orsynthetic polypeptide of claim 1, wherein the recombinant or syntheticpolypeptide induces an immune response in a mammal against at least onedengue virus of at least one serotype selected from the group ofdengue-1, dengue-2, dengue-3, and dengue-4.
 5. The recombinant orsynthetic polypeptide of claim 4, wherein the recombinant or syntheticpolypeptide induces an immune response in a mammal against at least onedengue virus of each of at least two serotypes selected from the groupof dengue-1, dengue-2, dengue-3, and dengue-4.
 6. The recombinant orsynthetic polypeptide of claim 5, wherein the recombinant or syntheticpolypeptide induces an immune response in a mammal against at least onedengue virus of each of at least three serotypes selected from the groupof dengue-1, dengue-2, dengue-3, and dengue-4.
 7. The recombinant orsynthetic polypeptide of claim 6, wherein the recombinant or syntheticpolypeptide induces an immune response in a mammal against at least onedengue virus of each of dengue-1, dengue-2, dengue-3, and dengue-4. 8.The recombinant or synthetic polypeptide of claim 1, wherein therecombinant or synthetic polypeptide induces an immune response in amammal against at least one dengue virus of at least one serotypeselected from the group of dengue-1, dengue-2, dengue-3, and dengue-4that is about equal to or greater than an immune response induced in themammal against the at least one dengue virus of the at least oneserotype by a dengue virus antigen selected from the group of SEQ IDNOS:149-152.
 9. The recombinant or synthetic polypeptide of claim 1,wherein the recombinant or synthetic polypeptide comprises an amino acidsequence that has at least about 95% amino acid sequence identity to atleast one sequence selected from the group of SEQ ID NOS:66, 69, 89, 93,108, and
 110. 10. The recombinant or synthetic polypeptide of claim 9,wherein the recombinant or synthetic polypeptide comprises an amino acidsequence selected from the group of SEQ ID NOS:66, 69, 89, 93, 108, and110.
 11. The recombinant or synthetic polypeptide of claim 1, whereinthe recombinant or synthetic polypeptide induces production ofantibodies capable of binding to at least one flavivirus.
 12. Therecombinant or synthetic polypeptide of claim 1, wherein the recombinantor synthetic polypeptide induces production of antibodies capable ofbinding to a first dengue virus of a first serotype and a second denguevirus of a second serotype, wherein the first serotype is different fromthe second serotype.
 13. The recombinant or synthetic polypeptide ofclaim 1, wherein the recombinant or synthetic polypeptide inducesproduction of neutralizing antibodies against at least one dengue virusof at least one serotype selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4.
 14. The recombinant or synthetic polypeptide ofclaim 13, wherein the recombinant or synthetic polypeptide inducesneutralizing antibodies against at least one dengue virus of each of atleast two serotypes selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4.
 15. The recombinant or synthetic polypeptide ofclaim 14, wherein the recombinant or synthetic polypeptide inducesneutralizing antibodies against at least one dengue virus of each of atleast three serotypes selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4.
 16. The recombinant or synthetic polypeptide ofclaim 15, wherein the recombinant or synthetic polypeptide inducesneutralizing antibodies against at least one dengue virus of each ofdengue-1, dengue-2, dengue-3, and dengue-4.
 17. A recombinant orsynthetic nucleic acid comprising a polynucleotide sequence having atleast about 85% sequence identity to at least one sequence selected fromthe group of SEQ ID NOS:157, 159, 172, 185, 187, and 235, or acomplementary polynucleotide sequence thereof.
 18. The recombinant orsynthetic nucleic acid of claim 17, where the recombinant or syntheticnucleic acid comprises a polynucleotide sequence having at least about90% sequence identity to at least one sequence selected from the groupof SEQ ID NOS:157, 159, 172, 185, 187, and 235, or a complementarypolynucleotide sequence thereof.
 19. The recombinant or syntheticnucleic acid of claim 17, wherein said recombinant or synthetic nucleicacid encodes a polypeptide that induces an immune response in a subjectagainst a flavivirus.
 20. The recombinant or synthetic nucleic acid ofclaim 19, wherein said recombinant or synthetic nucleic acid encodes apolypeptide that induces an immune response against at least one denguevirus.
 21. The recombinant or synthetic nucleic acid of claim 20,wherein said recombinant or synthetic nucleic acid encodes a polypeptidethat induces an immune response in a mammal against at least one denguevirus.
 22. The recombinant or synthetic nucleic acid of claim 21,wherein said nucleic acid encodes a polypeptide that induces productionof neutralizing antibodies in a mammal against at least one dengue virusof at least one serotype selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4.
 23. The recombinant or synthetic nucleic acid ofclaim 22, wherein said nucleic acid encodes a polypeptide that inducesproduction of neutralizing antibodies in a mammal against at least onedengue virus of each of at least two serotypes selected from the groupof dengue-1, dengue-2, dengue-3, and dengue-4.
 24. The recombinant orsynthetic nucleic acid of claim 23, wherein said nucleic acid encodes apolypeptide that induces production of neutralizing antibodies in amammal against at least one dengue virus of each of at least threeserotypes selected from the group of dengue-1, dengue-2, dengue-3, anddengue-4.
 25. The recombinant or synthetic nucleic acid of claim 23,wherein said nucleic acid encodes a polypeptide that induces productionof neutralizing antibodies in a mammal against at least one dengue virusof each of dengue-1, dengue-2, dengue-3, and dengue-4.
 26. Therecombinant or synthetic nucleic acid of claim 19, where the recombinantor synthetic nucleic acid comprises a polynucleotide sequence selectedfrom the group of SEQ ID NOS:157, 159, 172, 185, 187, and 235, or acomplementary polynucleotide sequence thereof.
 27. A recombinant orsynthetic nucleic acid comprising a polynucleotide sequence encoding apolypeptide selected from the group of SEQ ID NOS:66, 69, 89, 93, 108,and 110, or a sequence complementary to said polynucleotide sequence.28. A recombinant or synthetic nucleic acid having at least about 85% or90% sequence identity to an RNA polynucleotide sequence, said RNApolynucleotide sequence comprising a DNA sequence selected from thegroup of SEQ ID NOS:157, 159, 172, 185, 187, and 235 in which all of thethymine nucleotide residues in said DNA sequence arc replaced withuracil nucleotide residues, or a complementary sequence thereof.
 29. Therecombinant or synthetic nucleic acid of claim 28, wherein saidrecombinant or synthetic nucleic acid encodes a polypeptide that inducesan immune response in a mammal.
 30. The recombinant or synthetic nucleicacid of claim 29, wherein said polypeptide induces production ofneutralizing antibodies against at least one dengue virus of at leastone serotype selected from the group of dengue-1, dengue-2, dengue-3,and dengue-4.
 31. A recombinant or synthetic polypeptide comprising anamino acid sequence that has at least about 90% amino acid sequenceidentity to at least one polypeptide sequence selected from the group ofSEQ ID NOS:139, 141, 142-146, 148, 236, 237, 239, 251, 343, and
 345. 32.The recombinant or synthetic polypeptide of claim 31, wherein therecombinant or synthetic polypeptide induces an immune response in asubject against at least one flavivirus.
 33. The recombinant orsynthetic polypeptide of claim 31, wherein the recombinant or syntheticpolypeptide induces an immune response in a subject against at least onedengue virus.
 34. The recombinant or synthetic polypeptide of claim 31,wherein the recombinant or synthetic polypeptide induces an immuneresponse in a mammal against at least one dengue virus of at least oneserotype selected from the group of dengue-1, dengue-2, dengue-3, anddengue-4.
 35. The recombinant or synthetic polypeptide of claim 34,wherein the recombinant or synthetic polypeptide induces an immuneresponse in a mammal against at least one dengue virus of each of atleast two serotypes selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4.
 36. The recombinant or synthetic polypeptide ofclaim 35, wherein the recombinant or synthetic polypeptide induces animmune response in a mammal against at least one dengue virus of each ofat least three serotypes selected from the group of dengue-1, dengue-2,dengue-3, and dengue-4.
 37. The recombinant or synthetic polypeptide ofclaim 36, wherein the recombinant or synthetic polypeptide induces animmune response in a mammal against at least one dengue virus of each ofdengue-1, dengue-2, dengue-3, and dengue-4.
 38. The recombinant orsynthetic polypeptide of claim 31, wherein the recombinant or syntheticpolypeptide induces an immune response in a mammal against at least onedengue virus of at least one serotype selected from the group ofdengue-1, dengue-2, dengue-3, and dengue-4 that is about equal to orgreater than an immune response induced in the mammal against the atleast one dengue virus of the at least one serotype by a dengue virusantigen selected from the group of SEQ ID NOS:227-230.
 39. Therecombinant or synthetic polypeptide of claim 31, wherein therecombinant or synthetic polypeptide comprises an amino acid sequencethat has at least about 95% amino acid sequence identity to at least oneamino acid sequence selected from the group of SEQ ID NOS:139, 141,142-146, 148, 236, 237, 239, 251, 343, and
 345. 40. The recombinant orsynthetic polypeptide of claim 39, wherein the recombinant or syntheticpolypeptide comprises an amino acid sequence selected from the group ofSEQ ID NOS:139, 141, 142-146, 148, 236, 237, 239, 251, 343, and
 345. 41.The recombinant or synthetic polypeptide of claim 31, wherein therecombinant or synthetic polypeptide induces production of antibodiescapable of binding to at least one flavivirus.
 42. The recombinant orsynthetic polypeptide of claim 41, wherein the recombinant or syntheticpolypeptide induces production of antibodies capable of binding to afirst dengue virus of a first serotype and a second dengue virus of asecond serotype, wherein the first serotype is different from the secondserotype.
 43. The recombinant or synthetic polypeptide of claim 31,wherein the recombinant or synthetic polypeptide induces production ofneutralizing antibodies against at least one dengue virus of at leastone serotype selected from the group of dengue-1, dengue-2, dengue-3,and dengue-4.
 44. The recombinant or synthetic polypeptide of claim 43,wherein the recombinant or synthetic polypeptide induces neutralizingantibodies against at least one dengue virus of each of at least twoserotypes selected from the group of dengue-1, dengue-2, dengue-3, anddengue-4.
 45. The recombinant or synthetic polypeptide of claim 44,wherein the recombinant or synthetic polypeptide induces neutralizingantibodies against at least one dengue virus of each of at least threeserotypes selected from the group of dengue-1, dengue-2, dengue-3, anddengue-4.
 46. The recombinant or synthetic polypeptide of claim 45,wherein the recombinant or synthetic polypeptide induces neutralizingantibodies against at least one dengue virus of each of dengue-1,dengue-2, dengue-3, and dengue-4.
 47. A recombinant or synthetic nucleicacid comprising a polynucleotide sequence having at least about 85%sequence identity to at least one nucleotide sequence selected from thegroup of SEQ ID NOS:202, 205-210, 255, 258, 259, 261, 342, and 344, or acomplementary polynucleotide sequence thereof.
 48. The recombinant orsynthetic nucleic acid of claim 47, where the recombinant or syntheticnucleic acid comprises a polynucleotide sequence having at least about90% sequence identity to at least one nucleotide sequence selected fromthe group of SEQ ID NOS:202, 205-210, 255, 258, 259, 261, 342, and 344,or a sequence complementary to said polynucleotide sequence.
 49. Therecombinant or synthetic nucleic acid of claim 47, where the recombinantor synthetic nucleic acid comprises a polynucleotide sequence having atleast about 95% sequence identity to at least one nucleotide sequenceselected from the group of SEQ ID NOS:202, 205-210, 255, 258, 259, 261,342, and 344, or a sequence complementary to said polynucleotidesequence.
 50. The recombinant or synthetic nucleic acid of claim 47,wherein said recombinant or synthetic nucleic acid encodes a polypeptidethat induces an immune response against a flavivirus.
 51. Therecombinant or synthetic nucleic acid of claim 50, wherein saidrecombinant or synthetic nucleic acid encodes a polypeptide that inducesan immune response in a mammal against at least one dengue virus. 52.The recombinant or synthetic nucleic acid of claim 51, wherein saidnucleic acid encodes a polypeptide that induces production ofneutralizing antibodies against at least one dengue virus of at leastone serotype selected from the group of dengue-1, dengue-2, dengue-3,and dengue-4.
 53. The recombinant or synthetic nucleic acid of claim 52,wherein said nucleic acid encodes a polypeptide that induces productionof neutralizing antibodies in a mammal against at least one dengue virusof each of at least two serotypes selected from the group of dengue-1,dengue-2, dengue-3, and dengue-4.
 54. The recombinant or syntheticnucleic acid of claim 53, wherein said nucleic acid encodes apolypeptide that induces production of neutralizing antibodies in amammal against at least one dengue virus of each of at least threeserotypes selected from the group of dengue-1, dengue-2, dengue-3, anddengue-4.
 55. The recombinant or synthetic nucleic acid of claim 54,wherein said nucleic acid encodes a polypeptide that induces productionof neutralizing antibodies in a mammal against at least one dengue virusof each of dengue-1, dengue-2, dengue-3, and dengue-4.
 56. Therecombinant or synthetic nucleic acid of claim 49, where the recombinantor synthetic nucleic acid comprises a polynucleotide sequence selectedfrom the group of SEQ ID NOS:202, 205-210, 255, 258, 259, 261, 342, and344, or a complementary polynucleotide sequence thereof.
 57. Arecombinant or synthetic nucleic acid comprising a polynucleotidesequence encoding a polypeptide of claim 31, or a complementarypolynucleotide sequence thereof.
 58. A recombinant or synthetic nucleicacid having at least about 85% or 90% sequence identity to an RNApolynucleotide sequence, said RNA polynucleotide sequence comprising aDNA sequence selected from the group of SEQ ID NOS:202, 205-210, 255,258, 259, 261, 342, and 344 in which all of the thymine nucleotideresidues in said DNA sequence are replaced with uracil nucleotideresidues, or a complementary sequence thereof.
 59. The recombinant orsynthetic nucleic acid of claim 58, wherein said recombinant orsynthetic nucleic acid encodes a polypeptide that induces an immuneresponse in a mammal.
 60. The recombinant or synthetic nucleic acid ofclaim 59, wherein said polypeptide induces production of neutralizingantibodies against at least one dengue virus of at least one serotypeselected from the group of dengue-1, dengue-2, dengue-3, and dengue-4.61. A composition comprising at least one recombinant or syntheticpolypeptide of claim 1 and an excipient or carrier.
 62. A compositioncomprising at least one recombinant or synthetic polypeptide of claim 31and an excipient or carrier.
 63. A composition comprising at least onerecombinant or synthetic nucleic acid of claim 17 and an excipient orcarrier.
 64. A composition comprising at least one recombinant orsynthetic nucleic acid of claim 47 and an excipient or carrier.
 65. Avector comprising a recombinant or synthetic nucleic acid of claim 17.66. The vector of claim 65, wherein the vector is a DNA vector.
 67. Thevector of claim 65, wherein the vector is a RNA vector.
 68. The vectorof claim 65, wherein the recombinant or synthetic nucleic acid isoperably linked to a promoter.
 69. The vector of claim 65, wherein thevector is an expression vector.
 70. The vector of claim 65, wherein theexpression vector comprises the vector shown in FIG.
 1. 71. The vectorof claim 65, wherein the vector comprises a plasmid, a cosmid, a phage,a linear expression element, a nucleic acid-protein conjugate, a virus,or virus-like particle.
 72. The vector of claim 65, wherein the vectoris a viral vector.
 73. The vector of claim 72, wherein the viral vectoris a replication-deficient targeted viral vector.
 74. The vector ofclaim 72, wherein the viral vector is selected from the group of aflaviviral vector, adenoviral vector, retroviral vector, papilloma viralvector, an adeno-associated viral vector, alphavirus vector,hepadnavirus vector, baculovirus and a herpes viral vector.
 75. Thevector of claim 72, wherein the vector is an attenuated viral vector.76. The vector of claim 74, wherein the vector is a flaviviral vector.77. The vector of claim 76, wherein the vector comprises the nucleicacid sequence of a flavivirus genome lacking at least the nucleotidesegment of the genome that encodes the flaviviral envelope protein. 78.The vector of claim 65, wherein the vector comprises the nucleic acidsequence of a flavivirus genome lacking at least the nucleotide segmentof the genome that encodes the flaviviral envelope protein and thenucleotide segment of the genome that encodes the last 15 amino acids ofthe C terminus of the prM protein.
 79. A vector comprising a recombinantor synthetic nucleic acid of claim
 47. 80. The vector of claim 79,wherein the vector is a DNA vector.
 81. The vector of claim 79, whereinthe vector is a RNA vector.
 82. The vector of claim 79, wherein therecombinant or synthetic nucleic acid is operably linked to a promoter.83. The vector of claim 79, wherein the vector is an expression vector.84. The vector of claim 83, wherein the expression vector comprises thevector shown in FIG.
 1. 85. The vector of claim 79, wherein the vectorcomprises a plasmid, a cosmid, a phage, a linear expression element, anucleic acid-protein conjugate, a virus, or virus-like particle.
 86. Thevector of claim 79, wherein the vector is a viral vector.
 87. The vectorof claim 86, wherein the viral vector is a replication-deficienttargeted viral vector.
 88. The vector of claim 86, wherein the viralvector is selected from the group of a flaviviral vector, adenoviralvector, retroviral vector, papilloma viral vector, an adeno-associatedviral vector, alphavirus vector, hepadnavirus vector, and a herpes viralvector.
 89. The vector of claim 86, wherein the vector is an attenuatedviral vector.
 90. The vector of claim 86, wherein the vector is aflaviviral vector.
 91. The vector of claim 90, wherein the vectorcomprises the nucleic acid sequence of a flavivirus genome lacking atleast the nucleotide segment of the genome that encodes the flaviviralenvelope protein.
 92. The vector of claim 91, wherein the vectorcomprises the nucleic acid sequence of a flavivirus genome lacking atleast the nucleotide segment of the genome that encodes the flaviviralenvelope protein and the nucleotide segment of the genome that encodesthe last 15 amino acids of the C terminus of the prM protein.
 93. Thevector of claim 76, wherein the flaviviral vector is a yellow fevervirus vector or a dengue virus vector.
 94. The vector of claim 90,wherein the flaviviral vector is a yellow fever virus vector or a denguevirus vector.
 95. The composition of claim 61, wherein the excipient orcarrier is a pharmaceutically acceptable excipient or pharmaceuticallyacceptable carrier.
 96. The composition of claim 63, wherein theexcipient or carrier is a pharmaceutically acceptable excipient orpharmaceutically acceptable carrier.
 97. A composition comprising atleast one vector of claim 65 and an excipient or carrier.
 98. Thecomposition of claim 97, wherein the vector is a viral vector and thecomposition comprises from about 1×10² to about 1×10⁸ viral vectorparticles/mL.
 99. The composition of claim 97, wherein the compositioncomprises from about 1 μg to about 10 mg of the viral vector.
 100. Thecomposition of claim 61, wherein the composition comprises at least onepharmaceutically acceptable excipient, at least one pharmaceuticallyacceptable buffer, at least one pharmaceutically acceptable adjuvant, atleast one pharmaceutically acceptable diluent, at least onepharmaceutically acceptable liposome, or any combination thereof.
 101. Avirus-like particle comprising at least one polypeptide of claim
 1. 102.A virus-like particle comprising at least one polypeptide of claim 31.103. An attenuated or replication deficient virus comprising at leastone polypeptide of claim
 1. 104. An attenuated or replication deficientvirus comprising at least one polypeptide of claim
 31. 105. A cellcomprising at least one recombinant or synthetic polypeptide of claim 1.106. A cell comprising at least one recombinant or synthetic polypeptideof claim
 31. 107. A cell comprising at least one recombinant orsynthetic nucleic acid of claim
 17. 108. A method of producingantibodies against at least one dengue virus of at least one serotypecomprising administering to a subject at least one recombinant orsynthetic polypeptide of claim 1 in an amount sufficient to induceproduction of said antibodies.
 109. The method of claim 108, wherein apopulation of neutralizing antibodies against at least one dengue virusselected of at least one serotype selected from the group of dengue-1,dengue-2, dengue-3, and dengue-4 is induced.
 110. A method of producingantibodies against at least one dengue virus of at least one serotypecomprising administering to a subject at least one recombinant orsynthetic polypeptide of claim 31 in an amount sufficient to induceantibody production.
 111. A method of producing antibodies against atleast one dengue virus of at least one serotype comprising administeringto a subject at least one recombinant or synthetic nucleic acid of claim17 in an amount sufficient to induce antibody production.
 112. Themethod of claim 111, wherein a population of neutralizing antibodiesagainst at least one dengue virus of at least one serotype selected fromthe group of dengue-1, dengue-2, dengue-3, and dengue-4 is induced. 113.A method of producing antibodies against at least one dengue virus of atleast one serotype comprising administering to a subject at least onerecombinant or synthetic nucleic acid of claim 47 or a polypeptide ofthe invention in an amount sufficient to induce antibody production.114. A method of inducing an immune response in a subject to at leastone dengue virus of at least one serotype comprising administering aneffective amount of a recombinant or synthetic nucleic acid of claim 47to the mammal.
 115. The method of claim 114, wherein the method furthercomprises providing a first repeat administration of the at least onerecombinant or synthetic polypeptide to the subject at least about 14days to about six months after the recombinant or synthetic polypeptideis initially administered and optionally providing a second repeatadministration of the recombinant or synthetic polypeptide after thefirst repeat administration and at least about one month to 12 monthsafter recombinant or synthetic polypeptide is initially administered.116. The vector of claim 65, wherein the vector acid further comprises apolynucleotide sequence encoding at least one cytokine, adjuvant,co-stimulatory molecule, heterologous antigen, or any combinationthereof.
 117. The vector of claim 79, wherein the vector acid furthercomprises a polynucleotide sequence encoding at least one cytokine,adjuvant, co-stimulatory molecule, heterologous antigen, or anycombination thereof.